LANTIBIOTICS AND USES THEREOF

Abstract

vides isolated lantibiotics that inhibit Gram negative and Gram positive microbes. The lantibiotic includes an amino acid sequence, wherein the amino acid sequence of the compound and the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:22 have at least 80% identity. The lantibiotics have the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe. The present invention also provides methods for making and using the lantibiotics.

Description

CONTINUING APPLICATION DATA

[0001]This application claims the benefit of U.S. Provisional Application Ser. No. 60/961,374, filed Jul. 20, 2007, which is incorporated by reference herein.

BACKGROUND

[0003]Recent molecular studies into the microbial diversity of the human intestine reveal a much greater diversity than previously recognized and very little is currently known of the contribution of individual groups to the human organism (Gill et al., 2006, Science, 312:1355-1359). One numerically dominant group of microbes, the bifidobacteria, is often suggested to be associated with good intestinal health given their overriding dominance in the feces of breast fed infants (Yoshioka et al., 1983, Pediatrics, 72:317-321). This phenomenon led to their discovery in 1899 by the pediatrician Henri Tissier and his subsequent use of these bacteria for the treatment of infantile diarrhea (Tissier, 1906, Crit Rev Soc Biol, 60:359-361). The proposed beneficial effect of bifidobacteria is further supported by the decrease of these bacteria in geriatric individuals and the concomitant increase of other microbial groups, most notably clostridia and E. coli (Mitsuoka et al., 1973, Zentralbl Bakteriol [Orig A], 223:333-342, Hopkins et al., 2001, Gut, 48:198-205, Ishibashi et al, 1997, Mal J Nutr, 3:149-159). This has led to the growing worldwide interest of including bifidobacteria in foods specifically for their potential intestinal health benefits (O'Sullivan, Primary Sources of Probiotic Cultures, In: Probiotics in food safety and human health. Edited by Goktepe et al., Boca Raton: Taylor & Francis/CRC Press, 2006:91-107). However, clinical feeding studies with bifidobacteria show that while the strains can be detected in subject's feces during feeding trials, they are rapidly lost upon cessation of the studies pointing to a possible loss of competitive fitness of the strains for competition within the human intestinal environment (O'Sullivan, Primary Sources of Probiotic Cultures, In: Probiotics in food safety and human health. Edited by Goktepe et al., Boca Raton: Taylor & Francis/CRC Press, 2006:91-107, Fukushima et al., 1998, Int J Food Microbiol, 42:39-44, Su et al, 2005, FEMS Microbiol Lett, 244:99-103. This may be due to attenuation of the strains, as the fermentation environment is very different to the buffered and anaerobic environment of the human colon.

[0004]Bacteriocins are peptide based antimicrobial compounds produced by many types of bacteria and are inhibitory to closely related bacteria. Frequently, the inhibitory spectrum is within the genus of the producing bacterium. A lantibiotic is a type of bacteriocin that has a wide inhibitory spectrum and is also post-translationly modified. Specifically, modification enzymes modify some amino acids into lantionine residues. Nisin, which is produced by certain strains of the lactic acid bacterium Lactococcus lactis, is a lantibiotic with the widest inhibitory spectrum of any lantibiotic described to date that extends to most gram positive bacteria. Given its broad spectrum it is widely used as a preservative and a shelf life extender. Unfortunately, spoilage and pathogenic bacteria are not just gram positive. Many pathogens, such as E. coli and Salmonella are gram negative and many spoilage bacteria are also gram negative, such as Pseudomonas and Klebsiella.

SUMMARY OF THE INVENTION

[0005]The present invention provides a lantibiotic, entitled bisin, from a probiotic culture of Bifidobacterium longum that inhibits both gram positive and gram negative bacteria. This is the first bacteriocin described to date to have natural inhibitory action against both gram positive and gram negative bacteria. It therefore has potential to be an effective shelf life extender in dairy products, given that the enzymatic activities of gram negative bacteria, particularly Pseudomonas, are responsible for a lot of defects.

[0006]The potential to produce a lantibiotic was first recognized from the genome sequence of the Bifidobacterium longum strain described herein; however, initial attempts at detecting a lantibiotic produced by the strain were unsuccessful. Further experiments were required before growth conditions were found that caused a lantibiotic to be produced. Subsequently, bioassays were used to test its spectrum of inhibition and clearly showed effective inhibition against both gram positive and gram negative indicators.

[0007]The present invention provides an isolated biologically active compound that includes an amino acid sequence, wherein the amino acid sequence of the compound and the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:22 have at least 80% identity. The polypeptide sequence may include at least one conservative substitution of the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:22. The compound has the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe. The Gram negative may be an E. coli, a Serratia proteus, or a Salmonella spp. In some aspects it is preferably not a P. aeruginaosa. The compound inhibits growth of a Gram positive microbe, such as a Lactobacillus spp., Lactococcus spp., a Streptococcus spp., a Staphylococcus spp., or a Bacillus spp. The compound may be produced by a Bifidobacterium. The present invention also includes a composition having the isolated biologically active compound and a food product, and a composition having the isolated biologically active compound and a pharmaceutically acceptable carrier.

[0008]The present invention also provides an isolated polynucleotide including: (a) a nucleotide sequence encoding a polypeptide, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:22 have at least 80% identity, or (b) the full complement of the nucleotide sequence of (a). The isolated polynucleotide may be operably linked to a heterologous regulatory sequence. The present invention also provides a vector containing the isolated polynucleotide, and a cell containing the isolated polynucleotide.

[0009]The present invention further provides isolated lantibiotic, wherein the lantibiotic inhibits growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe. The lantibiotic may include an amino acid sequence, wherein the amino acid sequence of the compound and the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:22 have at least 80% identity. The Gram negative may be an E. coli, a Serratia proteus, or a Salmonella spp. In some aspects it is preferably not a P. aeruginaosa.

[0010]The present invention provides a composition with a lantibiotic and a food product, wherein the lantibiotic has the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe. The lantibiotic may be present on the surface of the food product, in the food product, or the combination. The lantibiotic may include an amino acid sequence, wherein the amino acid sequence of the lantibiotic and the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:22 have at least 80% identity. The Gram negative may be an E. coli, a Serratia proteus, or a Salmonella spp.

[0011]The present invention provides a composition with a lantibiotic and a pharmaceutically acceptable carrier, wherein the lantibiotic includes the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe. The lantibiotic may include an amino acid sequence, wherein the amino acid sequence of the lantibiotic and the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:22 have at least 80% identity. The Gram negative may be an E. coli, a Serratia proteus, or a Salmonella spp.

[0012]The present invention also provides methods for producing the compounds described herein. The methods may include growing an isolated Bifidobacterium under conditions suitable for producing a lantibiotic, wherein the Bifidobacterium produces a lantibiotic. The method may further include isolating the lantibiotic. The growing may include growing the Bifidobacterium, preferably B. longum, on a surface. The present invention also includes a lantibiotic produced by the method.

[0013]Method for producing a lantibiotic may include growing a microbe that includes a polynucleotide encoding a polypeptide, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:22 have at least 80% identity, wherein the microbe is grown under conditions suitable for producing the polypeptide, and wherein the microbe produces the polypeptide. The microbe may further include a polynucleotide encoding a polypeptide selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or a combination thereof. The growing may include growing the Bifidobacterium, preferably B. longum, on a surface. The present invention also includes a polypeptide produced by the method. The method may further include isolating the polypeptide, for instance by extraction with a composition that includes an alcohol, such as methanol.

[0014]Further provided by the present invention are methods for using lantibiotics. A method may include adding the lantibiotic to a food product, wherein the lantibiotic includes the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe. The method may include applying the lantibiotic to the surface of the food product, for instance, by bringing a surface of a casing, film, or packaging material comprising the lantibiotic into contact with the food product. The adding may include adding the lantibiotic to the food product. The lantibiotic may act as a food preservative.

[0015]The present invention provides a dentifrice, such as a mouthwash or a toothpaste, that includes a biologically active compound with an amino acid sequence, wherein the amino acid sequence of the compound and the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:22 have at least 80% identity, wherein the compound has the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe.

[0016]Also provided by the present invention is a method for using a lantibiotic that includes administering a composition with the lantibiotic to an animal, such as a human, wherein the subject has or is at risk of an infection by a microbe that is inhibited by the lantibiotic, and wherein the lantibiotic has the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe. The composition may include a pharmaceutically acceptable carrier, and the composition may be administered topically.

[0017]The present invention also provides isolated biologically active polypeptides, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, have at least 80% identity. Also included in the invention are isolated polynucleotides encoding the polypeptides.

[0018]The present invention further provides a Bifidobacterium that produces a lantibiotic. The lantibiotic has the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe. The Gram negative may be an E. coli, a Serratia proteus, or a Salmonella spp. The Bifidobacterium may be encapsulated or in tablet form, for instance, and may be present in a food product. The present invention also provides method that includes administering a Bifidobacterium to an animal in need thereof, wherein the Bifidobacterium will produce a lantibiotic that has the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe.

[0019]The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0020]The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0021]The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

[0022]Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0023]Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0024]For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0025]The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE FIGURES

[0026]FIG. 1. Organization of mobile integrase cassettes (MIC) in B. longum DJO10A. (A) and NCC2705, (B). Orfs 1, 2 and 3 refer to three contiguous, but different xerC integrase genes. P, a conserved 20 bp palindrome (TTAAACCGACATCGGTTTAA (SEQ ID NO:24), which has an 11 bp extension in MIC III. IR, 96 bp inverted repeat (IR) (GATTAAGCCGGGTTTGTTGTTAAGCCGGGGAACGGTTCGGGGTCTTGGT GGCTGGCCGTGTCCCATGTGGTTTCCCGGCTTAACGTTCCGGGTTAT (SEQ ID NO:25)), that has a 3 bp extension in MIC I and II, a 5 bp extension in MIC III and a 1 bp extension in MIC 1, 2 and 3. IS, insertion sequence.

[0027]FIG. 2. Genome unique regions. (A) Base deviation index (BDI) analysis of the B. longum DJO10A and NCC2705 genomes. Unique regions of each genome as defined in the text are numbered. The locations of oriC and terC are indicated by green arrows. Letters refer to predicted gene phenotypes from regions with definitive BDI peaks that are present in both genomes, a, GTPase, b, cation transport ATPase, c, DNA partitioning protein, d, choloylglycine hydrolase, e, glutamine synthase beta chain, f, alanyl-tRNA synthetase, g, pyruvate kinase, h, cation transport ATPase, I, fibronectin type III, j, aminopeptidase C, k, subtilisin-like serine protease, 1, sortase, m, fatty acid synthase. (B) Organization of the unique region 1 showing the location of a 361 bp DNA remnant, indicated by the green bar, from the ushA gene remaining at the predicted deletion location in NCC2705. Sky blue colored ORFs indicate common genes between both genomes. a, mobile integrase cassette.

[0028]FIG. 3. Comparison of oligosaccharide utilization gene cluster 7 between two B. longum genomes. DJO10A-unique genes in unique region 10 are colored dark grey, ISL3-type IS element is colored black and other matched genes are colored white. galA, α-galactosidase; lacI, LacI-type repressor; malEFG, ABC-type transport system; ISL3, ISL3-type IS element; agl1, glycosidase; ilvA, threonine dehydratase; SIR2, NAD-dependent protein deacetylase; glyH, glycosyl hydrolase; hyp, hypothetical protein.

[0029]FIG. 4. Organization of genes involved in polyol metabolism in the unique region 13 in strain DJO10A and comparison with an analogous region in B. adolescentis ATCC 15703. Amino acid identities are indicated between homologous genes. ORFs shaded black are from unique region 13 and corresponding homologs in B. adolescentis ATCC 15703.

[0030]FIG. 5. Arsenic resistance of selected bacteria. (A) Genetic organization of arsenic resistance gene clusters compiled from the completed genome sequences of Bifidobacterium longum DJO10A, Bacillus subtilis 168 (Kunst et al., 1997, Nature 1997, 390:249-256), Bacteroides thetaiotamicron VPI-5482 (Xu et al., 2003, Science 2003, 299:2074-2076), Lactobacillus brevis ATCC 367 (Makarova et al., Proc Natl Acad Sci USA 103:15611-15616), L. plantarum WCFS1 (Kleerebezem et al., 2003, Proc Natl Acad Sci USA 2003, 100:1990-1995), L. johnsonii NCC 533 (Pridmore et al., 2004, Proc Natl Acad Sci USA 2004, 101:2512-2517) and E. coli K-12 (Sofia et al., 1994, Nucleic Acids Res 1994, 22:2576-2586). a, 48 kb element that is excised by the site-specific recombinase SpoIVCA during sporulation, b, indicates a plasmid sequence, arsR, repressor, arsA, arsenite stimulated ATPase, arsB, arsenite efflux pump, arsC, arsenate reductase, arsD, arsenic chaperone, hyp, hypothetical protein. (B) Comparison of arsenic resistance activity in B. longum DJO10A with fermentation adapted B. animalis subsp. lactis strains, E. coli and Lactobacillus plantarum. c, calculated from data presented in van Kranenburg et al., (van Kranenburg et al., 2005, Appl Environ Microbiol 2005, 71:1223-1230).

[0031]FIG. 6. Lantibiotic production by B. longum DJO10A. (A) Organization of the lantibiotic encoding unique region 12 of B. longum DJO10A and the corresponding genome locations in strains NCC2705 and DJO10A-JH1. The A or B designator following IS30 refer to unique classes of IS30 elements that are only found at this location in the genome. The 'designator indicates a fragmented IS30 element. (B) Pulsed Field Gel Electrophoresis (PFGE) analysis of XbaI-digested total DNA from B. longum DJO10A and its fermentation adapted isolate, DJO10A-JH1. White arrows indicate bands missing from strain DJO10A-JH1. (C) Bioassay for lantibiotic production by B. longum DJO10A with strains DJO10A and DJO10A-JH1 as indicator bacteria.

[0032]FIG. 7. IS30 `jumping` in the genome of B. longum DJO10A. (A) Genome positioning of the IS30 elements in the genome of B. longum DJO10A and the laboratory adapted strain DJO10A-JH1. The gray arrows indicate the five elements identified by direct sequencing of DJO10A genomic DNA. The white arrows indicate the location of elements that were detected in some sequencing clones prepared from DJO10A genomic DNA. The asterisk under A6 indicates this element was missing from some sequencing clones of DJO10A DNA. (B) NruI digested genomic DNA from DJO10A shown in the left gel and its Southern hybridization (right gel) using probes specific for four different IS element families. (1) refers to DJO10A and (2) refers to DJO10A-JH1. Arrows indicate bands in DJO10A corresponding to specific IS30 elements as illustrated in (A).

[0033]FIG. 8. Simulated fecal competitive analysis of B. longum DJO10A and its in vitro adapted derivative, strain DJO10A-JH1, against Clostridium difficile and E. coli. (A) Viable cell counts of E. coli DJOec1 at the beginning of the competitive study (black), following competition with B. longum DJO10A-JH1 (horizontal lines) and B. longum DJO10A (hatched). (B) Viable cell counts of C. difficile DJOcd1 at the beginning of the competitive study (black), following competition with B. longum DJO10A-JH1 (horizontal lines) and B. longum DJO10A (hatched). N=3.

[0034]FIG. 9. Conserved structure of the oriC region. This consists of three clusters, in the two B. longum genomes. The DnaA boxes consist of 7 types, designated A to G as follows: Type A (TTATCCACA), Type B (TTGTCCACA), Type C (TTTTCCACA), Type D (TTACCCACA), Type E (TTATCCACC), Type F (TTATTCACA), Type G (TTATGCACA).

[0035]FIG. 10. Type I and II restriction modification (R-M) systems encoded by the B. longum genomes. (A) Alignment of the genomic locations encoding a type I R-M system between B. longum DJO10 and NCC2705. (B) Comparison of a Sau3AI-type II R-M system (recognition site, 5'-GATC-3') with analogous R-M systems in other bacteria and (C) comparison of a EcoRII-type II R-M system (recognition site, 5'-CCWGG-3') with analogous R-M systems in other bacteria. Percentage protein sequence identities compared to B. longum DJO10A are indicated.

[0036]FIG. 11. Organization of the 11 different types of oligosaccharide utilization gene clusters (11 in DJO10A and 7 in NCC2705). Unique genes of strain DJO10A are indicated. IS, insertion sequence; Hyp, hypothetical protein; Arab, arabinosidase; E, malE; F, malF; G,malG; R, lacI-type repressor; K, ATPase of ABC transporter; αGal, α-galactosidase; βXy1, β-xylosidase; Est, esterase; LCFACS, long-chain fatty acid acetyl CoA synthetase; f, fragmented gene; Xy1T, D-xylose proton symporter; βGal, β-galactosidase; Arab-βGal, arabinogalactan endo-1,4-β-galactosidase; O157, ORF with homolog only in E. coli O157; αMan, α-mannosidase; GlycH, glycosyl hydrolase; NAc-Glc, N-acetyl glucosaminidase; UhpB, histidine kinase; RfbA, dTDP-glucose pyrophosphorylase; RfbB, dTDP-D-glucose 4,6-dehydratase; RfbC, dTDP-4-dehydrorhamnose 3,5-epimerase; RgpF, lipopolysaccharide biosynthesis protein; TagG, ABC-type polysaccharide/polyol phosphate export systems, permease component; TagH, ABC-type polysaccharide/polyol phosphate transport system, ATPase component; MdoB, phosphoglycerol transferase; ProP, permease; Acyl-Est, acyl esterase. It should be noted that the glycosyl hydrolase gene in cluster 7 was annotated as isomaltase in the NCC2705 genome annotation.

[0037]FIG. 12. Nucleotide substitution analysis of all gene homologs between B. longum DJO10A and NCC2705, according to the dN:dS ratio.

[0038]FIG. 13. Organization of four predicted LPXTG-type, cell surface anchor proteins in B. longum DJO10A. The numbers below the signal peptide boxes indicate the location of signal peptides. The size of the respective proteins is indicated in amino acids.

[0039]FIG. 14. Loss of the lantibiotic gene cluster from B. longum DJO10A-JH1. (A) Detection of DJO10A specific gene clusters in B. longum DJO10A and its fermentation adapted isolate DJO10A-JH1 by PCR. M, 1 kb DNA ladder (Invitrogen); lane 1, unique region 15; lane 2, unique region 6; lane 3, unique region 9; lane 4, unique region 11; lane 5, unique region 5; lane 6, unique region 7; lane 7, unique region 12; lane 8, 16S rRNA partial gene. The arrow indicates the lantibiotic encoded unique region 12 that is missing from strain DJO10A-JH1. (B) Southern blot analysis using a lanM probe and the EcoRI-digested genomes of B. longum strains DJO10A and DJO10A-JH1. The 1.7 kb EcoRI band containing lanM is indicated with an arrow.

[0040]FIG. 15. Growth curves in RCM medium of the four bacteria used in the fecal competitive growth experiments. All bacteria were inoculated at 1% from freshly grown cultures. Squares, E. coli DJOec1; triangles, Clostridium difficile DJOcd1; circles, B. longum DJO10A-JH1; and diamonds, B. longum DJO10A.

[0041]FIG. 16. Portion of genomic sequence of B. longum DJO10A (Genbank Accession No. CP000605) including the lantibiotic-encoding gene cluster (SEQ ID NO:23). Present within SEQ ID NO:23: nucleotides 1979049-1979753 (SEQ ID NO:1) and the polypeptide encoded thereby (SEQ ID NO:2); nucleotides 1979747-1980907 (SEQ ID NO:3) and the polypeptide encoded thereby (SEQ ID NO:4); nucleotides 1981217-1981417 (SEQ ID NO:5) and the polypeptide encoded thereby (SEQ ID NO:6); nucleotides 1981501-1982160 (SEQ ID NO:7) and the polypeptide encoded thereby (SEQ ID NO:8); nucleotides 1982200-1982937 (SEQ ID NO:9) and the polypeptide encoded thereby (SEQ ID NO:10); nucleotides 1983009-1986110 (SEQ ID NO:11) and the polypeptide encoded thereby (SEQ ID NO:12); nucleotides 1986161-1986979 (SEQ ID NO:13) and the polypeptide encoded thereby (SEQ ID NO:14); nucleotides 1986976-1989213 (SEQ ID NO:15) and the polypeptide encoded thereby (SEQ ID NO:16).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0042]The present invention provides compounds that inhibit the growth of certain microbes. A compound of the present invention includes a polypeptide. As used herein, the term "polypeptide" refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term "polypeptide" also includes molecules which contain more than one polypeptide joined by a disulfide bond, or complexes of polypeptides that are joined together, covalently or noncovalently, as multimers (e.g., dimers, tetramers). Thus, the terms peptide, oligopeptide, and protein are all included within the definition of polypeptide and these terms are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. A compound of the present invention can be referred to herein as a lantibiotic. Preferably, a compound of the present invention is isolated. As used herein, an "isolated" polypeptide, such as a lantibiotic, or polynucleotide refers to a polypeptide or polynucleotide that has been either removed from its natural environment, produced using recombinant techniques, or chemically or enzymatically synthesized. Preferably, a polypeptide or polynucleotide of this invention is purified, i.e., essentially free from any other polypeptide or polynucleotide and associated cellular products or other impurities.

[0043]Without intending to be limiting, during production of a compound of the present invention by a microbe, such as a Bifidobacterium, a prepeptide is produced and subsequently processed in three steps; dehydration of certain amino acids, formation of thioether linkages between certain amino acids, and cleavage by a signal peptidase. The initial prepeptide may have the amino acid sequence SEQ ID NO:6.

[0044]The prepeptide is processed by dehydration to result in an intermediate. Serine residues may be dehydrated to form didehydroalanine. Thus, with reference to SEQ ID NO:6, the serine amino acids at position 36, 38, 42, 45, 47, 49, 52, 61 or a combination thereof, may be dehydrated to form didehydroalanine. Preferably, the serine amino acids at positions 47, 49, and 61 are dehydrated to form didehydroalanine. Threonine amino acids may be dehydrated to form didehydrobutyrine. Thus, with reference to SEQ ID NO:6, the threonine amino acids at positions 54, 57 or a combination thereof, may be dehydrated to form didehydrobutyrine. Preferably, the threonines at both positions 54 and 57 are dehydrated to form didehydrobutyrine.

[0045]Thus, an intermediate polypeptide resulting from the dehydration amino acids may have the following structure:

Methionine-Serine-Isoleucine-Aspartic acid-Glutamic acid-Lysine-Serine-Isoleucine-Valine-Glycine-Glutamic acid-Serine-Phenylalanine-Glutamic acid-Aspartic acid-Leucine-Serine-Alanine-Alanine-Aspartic acid-Methionine-Alanine-Methionine-Leucine-Threonine-Glycine-Arginine-Asp- aragine-Aspartic acid-Aspartic acid-Glycine-Valine-Alanine-proline-alanine-Xaa1-leucine-Xaa2-phenylalani- ne-alanine-valine-Xaa3-valine-leucine-Xaa4-valine-Xaa5-phenylalanine-Xaa6-- alanine-cysteine-Xaa7-valine-Xaa8-valine-valine-Xaa9-arginine-leucine-alan- ine-Xaa10-cysteine-glycine-asparagine-cysteine-lysine (SEQ ID NO:19)where Xaa1, 2, 3, 4, 5, 6, 7, and 10 are each independently serine or didehydroalanine; and Xaa8 and 9 are each independently threonine or didehydrobutyrine. A preferred example of an intermediate polypeptide resulting from the dehydration of certain amino acids isMethionine-Serine-Isoleucine-Aspargine-Glutamic acid-Lysine-Serine-Isoleucine-Valline-Glycine-Glutamic acid-Serine-Phenylalanine-Glutamic acid-Aspartic acid-Leucine-Serine-Alanine-Alanine-Aspartic acid-Methionine-Alanine-Methionine-Leucine-Threonine-Glycine-Arginine-Asp- aragine-Aspartic acid-Aspartic acid-Glycine-Valine-Alanine-proline-alanine-serine-leucine-serine-phenyla- lanine-alanine-valine-serine-valine-leucine-serine-valine-didehydroalanine- -phenylalanine-didehydroalanine-alanine-cysteine-serine-valine-didehydrobu- tyrine-valine-valine-didehydrobutyrine-arginine-leucine-alanine-didehydroa- lanine-cysteine-glycine-asparagine-cysteine-lysine (SEQ ID NO:20).

[0046]The polypeptide resulting from the dehydration of certain amino acids is further processed to form thioether linkages between certain amino acids. Didehydrobutyrine residues may be processed to form 2-aminobutyric acid (Abu) when used to form a thioether linkage with another amino acid, didehydroalanine residues may be processed to form alanine when used to form a thioether linkage with another amino acid, and cysteine residues may be processed to form alanine when used to form a thioether linkage with another amino acid. As has been observed in lantibiotics, lanthionine and 3-methyllanthionine residues result from the formation of thioether linkages between different amino acids.

[0047]The processed polypeptide is further processed by cleavage between two amino acids. The expected cleavage site is between amino acids 33 and 34. Other cleavage sites in SEQ ID NO:6 may be used to result in a processed peptide.

[0048]Thus, a compound of the present invention may have the following sequence:

proline-alanine-Xaa1-leucine-Xaa2-phenylalanine-alanine-valine-Xaa3-valine- -leucine-Xaa4-valine-Xaa5-phenylalanine-Xaa6-alanine-Xaa7-Xaa8-valine-Xaa9- -valine-valine-Xaa10-arginine-leucine-alanine-Xaa11-Xaa12-glycine-asparagi- ne-Xaa13-lysine (SEQ ID NO:21)where Xaa1, 2, 3, 4, 5, 6, 8, and 11 are each independently serine, didehydroalanine, or alanine; Xaa7, 12, and 13 are each independently cysteine or alanine, and Xaa9 and 10 are each independently threonine, didehydrobutyrine, or 2-aminobutyric acid. A preferred example of a compound of the present invention isproline-alanine-serine-leucine-serine-phenylalanine-alanine-valine-seri- ne-valine-leucine-serine-valine-alanine-phenylalanine-didehydroalanine-ala- nine-alanine-serine-valine-didehydrobutyrine-valine-valine-2-aminobutyric acid-arginine-leucine-alanine-alanine-alanine-glycine-asparagine-alanine-- lysine (SEQ ID NO:22).

[0049]A compound may have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6 thioether cross links. Preferably, a compound of the present invention has at least 1, more preferably at least 2, most preferably 3 cross links. The cross links can be between any two residues at positions Xaa 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 of SEQ ID NO:21, in any combination. Preferably, each cross link includes one cysteine residue, i.e., Xaa 7, 12, or 13 of SEQ ID NO:21. A preferred example of a compound of the present invention is SEQ ID NO:22 with thioether cross links between the amino acids at positions 14 (Xaa 5) and 18 (Xaa 7), positions 24 (Xaa 10) and 29 (Xaa 12), and positions 28 (Xaa 11) and 32 (Xaa 13).

[0050]A compound of the present invention may include polypeptides other than those depicted at SEQ ID NOs: 19, 20, 21, or 22, preferably, SEQ ID NO:21. For instance, a compound of the present invention may include those having structural similarity with another amino acid sequence. The similarity is referred to as structural similarity and is generally determined by aligning the residues of the two amino acid sequences (i.e., a candidate amino acid sequence and the amino acid sequence of SEQ ID NOs: 19, 20, 21, or 22) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A candidate amino acid sequence is the amino acid sequence being compared to an amino acid sequence present in an amino acid sequence, such as SEQ ID NO:21. A candidate amino acid sequence may be isolated from a Bifidobacterium, or may be produced using recombinant techniques, or chemically or enzymatically synthesized. Preferably, two amino acid sequences are compared using the BESTFIT algorithm in the GCG package (version 10.2, Madison Wis.), or the Blastp program of the BLAST 2 search algorithm, as described by Tatusova, et al. (FEMS Microbiol Lett 1999, 174:247-250), and available through the World Wide Web, for instance at the internet site maintained by the National Center for Biotechnology Information, National Institutes of Health. Preferably, the default values for all BLAST 2 search parameters are used, including matrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gap x_dropoff=50, expect=10, wordsize=3, and optionally, filter on. In the comparison of two amino acid sequences using the BLAST search algorithm, structural similarity is referred to as "identities." Preferably, a compound of the present invention also includes polypeptides with an amino acid sequence having at least 80% amino acid identity, at least 81% amino acid identity, at least 82% amino acid identity, at least 83% amino acid identity, at least 84% amino acid identity, at least 85% amino acid identity, at least 86% amino acid identity, at least 87% amino acid identity, at least 88% amino acid identity, at least 89% amino acid identity, at least 90% amino acid identity, at least 91% amino acid identity, at least 92% amino acid identity, at least 93% amino acid identity, at least 94% amino acid identity, at least 95% amino acid identity, at least 96% amino acid identity, at least 97% amino acid identity, at least 98% amino acid identity, or at least 99% amino acid identity to SEQ ID NOs: 19, 20, 21, or 22, preferably SEQ ID NO:21.

[0051]A compound of the present invention having structural similarity to SEQ ID NOs: 19, 20, 21, or 22, preferably SEQ ID NO:21, may include one or more conservative substitutions of the sequence disclosed at SEQ ID NOs: 19, 20, 21, or 22. A conservative substitution is typically the substitution of one amino acid for another that is a member of the same class. For example, it is well known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity, and/or hydrophilicity) can generally be substituted for another amino acid without substantially altering the secondary and/or tertiary structure of a polypeptide. For the purposes of this invention, conservative amino acid substitutions are defined to result from exchange of amino acids residues from within one of the following classes of residues: Class I: Gly, Ala, Val, Leu, and Ile (representing aliphatic side chains); Class II: Gly, Ala, Val, Leu, Ile, Ser, and Thr (representing aliphatic and aliphatic hydroxyl side chains); Class III: Tyr, Ser, and Thr (representing hydroxyl side chains); Class IV: Cys and Met (representing sulfur-containing side chains); Class V: Glu, Asp, Asn and Gln (carboxyl or amide group containing side chains); Class VI: His, Arg and Lys (representing basic side chains); Class VII: Gly, Ala, Pro, Trp, Tyr, Ile, Val, Leu, Phe and Met (representing hydrophobic side chains); Class VIII: Phe, Trp, and Tyr (representing aromatic side chains); and Class IX: Asn and Gln (representing amide side chains). The classes are not limited to naturally occurring amino acids, including amino acids not coded for in the standard genetic code and resulting from, for instance, post-translational modification of an amino acid, but also include artificial amino acids. A conservative substitution may be present at any location, preferably, at position 1, 2, 4, 6, 7, 8, 10, 11, 13, 15, 17, 20, 22, 23, 25, 26, 27, 30, 31, 33, or a combination thereof of SEQ ID NO:21.

[0052]Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al. (1990, Science, 247:1306-1310), wherein the authors indicate proteins are surprisingly tolerant of amino acid substitutions. For example, Bowie et al. disclose that there are two main approaches for studying the tolerance of a polypeptide sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selects or screens to identify sequences that maintain functionality. As stated by the authors, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require non-polar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie et al, and the references cited therein.

[0053]Preferably, a compound of the present invention is biologically active. As used herein, a "biologically active" compound or a compound having "biological activity" is one that inhibits growth of an indicator microbe. When the lantibiotic to be tested for biological activity is being produced by a microbe, preferably a Bifidobacterium, the microbe may be used to inoculate the center of an agar plate and incubated for a period of time, for instance, 2 days, to allow for replication of the microbe and production of the lantibiotic. Preferably, the agar plate is MRS or BLIM. Next, an indicator strain, previously grown on a different plate or in broth, is suspended in a molten top agar, for instance, 0.5% agar, and poured over the plate that contains the microbe producing the lantibiotic to be tested. The amount of indicator strain used can vary, but is typically added at a concentration that will yield visible growth in 1 to 2 days in the absence of a lantibiotic-producing microbe. The top agar is allowed to cool and harden, and the plate is incubated under conditions to allow growth of the indicator strain. The absence of the indicator strain around the microbe inoculated in the middle of the plate indicates the microbe is producing a lantibiotic with biological activity. The plate may be completely devoid of growth of the indicator strain, or there may be a halo of no indicator strain in the center of the plate.

[0054]When the lantibiotic to be tested for biological activity is isolated or purified, a hole may be cut in the center of an agar plate, and a solution containing an isolated or purified lantibiotic may be added to the hole and allowed to diffuse into the agar. Next, an indicator strain, previously grown on a different plate or in broth, is suspended in a molten top agar, for instance, 0.5% agar, and poured over the plate that contains the isolated or purified lantibiotic. The top agar is allowed to cool and harden, and the plate is incubated under conditions to allow growth of the indicator strain. The absence of the indicator strain around the microbe inoculated in the middle of the plate indicates the microbe is producing a lantibiotic with biological activity. The plate may be completely devoid of growth of the indicator strain, or there may be a halo of no indicator strain in the center of the plate.

[0055]Some lantibiotics are known to have some biological activity against gram negative microbes, but typically the biological activity exists only if the outer membrane of the gram negative microbe is damaged before exposure to the lantibiotic. The lantibiotic of the present invention has biological activity against gram negative microbes in the absence of damage to the outer membrane. Accordingly, testing whether a lantibiotic has biological activity is preferably done under conditions that do not damage the outer membrane of a gram negative microbe. Such conditions include, for instance, inclusion of a chelator in the medium, subjecting the indicator microbe to conditions of osmotic shock, heat, hydrostatic pressure, exposure to sub-lethal antimicrobials that effect the lipopolysaccarhide of the outer membrane, sub-lethal microwave exposure, and sublethal sonication. Likewise, some lantibiotics are known to have some biological activity against gram negative microbes, but typically the biological activity exists only if the gram negative microbe is exposed to higher concentrations of the lantibiotic than are used to inhibit a gram positive microbe (Hillman, U.S. Patent Application 20020128186. The lantibiotic of the present invention has biological activity against gram negative microbes and gram positive microbes when used at the same concentration.

[0056]Preferred indicator strains include, for instance, Micrococcus letteus, Lactococcus lactis, Staphylococcus aureus, Staphylococcus epidermdis, E. coli, Serratia marcescens, and Proteus vulgaris.

[0057]A compound of the present invention also has the characteristics of being resistant to heating to 100° C. for 10 minutes, inactivated by proteolytic digestion with pepsin at pH 2 and pronase E at pH7.5, and not inactivated by proteolytic digestion with α-Chymotrypsin, proteinase K, trypsin, and thermolysin. The compound is predicted to have an isoelectric point of 9.5, and a molecular weight of 3291.8 Daltons.

[0058]The present invention also provides other isolated polypeptides. Without intending to be limiting, production of a compound of the present invention, for instance, SEQ ID NO:22, by a microbe such as a Bifidobacterium, is facilitated by 7 other polypeptides. The naturally occurring versions of these 7 polypeptides are encoded by a set of coding regions including the coding region encoding the naturally occurring preprotein SEQ ID NO:6, and the expression of each of these 7 polypeptide results in the production of a compound, for instance, SEQ ID NO:22.

[0059]These 7 polypeptides are a response regulator of two component system (SEQ ID NO:2), a signal transduction histidine kinase (SEQ ID NO:4), a response regulator (SEQ ID NO:8), a prepeptide modification polypeptide (SEQ ID NO:10), a modifying enzyme (SEQ ID NO:12), an immunity polypeptide (SEQ ID NO:14), and a transporter polypeptide (SEQ ID NO:16). The transporter polypeptide is predicted to include protease capability to cleave the prepeptide. Also included in the invention are polypeptides having structural similarity with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. The similarity is referred to as structural similarity and is generally determined by aligning the residues of the two amino acid sequences (i.e., a candidate amino acid sequence and the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16) as described above. Preferably, a polypeptide of this aspect of the invention also includes polypeptides with an amino acid sequence having at least 80% amino acid identity, at least 81% amino acid identity, at least 82% amino acid identity, at least 83% amino acid identity, at least 84% amino acid identity, at least 85% amino acid identity, at least 86% amino acid identity, at least 87% amino acid identity, at least 88% amino acid identity, at least 89% amino acid identity, at least 90% amino acid identity, at least 91% amino acid identity, at least 92% amino acid identity, at least 93% amino acid identity, at least 94% amino acid identity, at least 95% amino acid identity, at least 96% amino acid identity, at least 97% amino acid identity, at least 98% amino acid identity, or at least 99% amino acid identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16. A polypeptide the present invention having structural similarity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 may include one or more conservative substitutions of the sequence disclosed at SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16.

[0060]A polypeptide having structural similarity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 preferably has activity of producing a biologically active compound of SEQ ID NO:22. Whether a polypeptide having structural similarity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 has activity can be determined by expressing one of the polypeptides with an altered amino acid sequence in a microbe, preferably a Bifidobacterium, with the other naturally occurring polypeptides, and determining whether a biologically active compound of the present invention is produced. For instance, if a polypeptide having structural similarity to SEQ ID NO:2 is to be tested for activity, it may be expressed in a cell with SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16, and the cell grown under conditions suitable for the production of a compound of the present invention. If the cell produces a compound having biological activity, then the tested polypeptide, i.e., the polypeptide having structural similarity to SEQ ID NO:2, is active.

Polynucleotides

[0061]The present invention also provides polynucleotides, preferably isolated polynucleotides. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded DNA and RNA. A polynucleotide may include nucleotide sequences having different functions, including for instance coding sequences, and non-coding sequences such as regulatory sequences. Coding sequence, non-coding sequence, and regulatory sequence are defined below. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide can be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment.

[0062]One polynucleotide of the present invention includes SEQ ID NO:17 (nucleotides 1981316-1981417 of SEQ ID NO:23), which encodes the polypeptide depicted at amino acids 34-66 of SEQ ID NO:6. It should be understood that a polynucleotide encoding a polypeptide represented by amino acids 34-66 of SEQ ID NO:6 is not limited to the nucleotide sequence disclosed at SEQ ID NO:17, but also includes the class of polynucleotides encoding such a polypeptide as a result of the degeneracy of the genetic code. For example, the naturally occurring nucleotide sequence SEQ ID NO:17 is but one member of the class of nucleotide sequences encoding a polypeptide having the amino acid sequence depicted at amino acids 34-66 of SEQ ID NO:6.

[0063]Other polynucleotides encoding a biologically active polypeptide of the present invention include those having structural similarity with the nucleotide sequence of SEQ ID NO:17. The similarity is referred to as structural similarity and is determined by aligning the residues of the two polynucleotides (i.e., the nucleotide sequence of the candidate sequence and the nucleotide sequence of SEQ ID NO:17) to optimize the number of identical nucleotides along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of shared nucleotides, although the nucleotides in each sequence must nonetheless remain in their proper order. A candidate sequence is the sequence being compared to SEQ ID NO:17. A candidate nucleotide sequence may be isolated from a Bifidobacterium, or may be produced using recombinant techniques, or chemically or enzymatically synthesized. Preferably, two nucleotide sequences are compared using the BESTFIT algorithm in the GCG package (version 10.2, Madison Wis.), or the Blastn program of the BLAST 2 search algorithm, as described by Tatusova, et al. (FEMS Microbiol Lett 1999, 174:247-250), and available through the World Wide Web, for instance at the internet site maintained by the National Center for Biotechnology Information, National Institutes of Health. Preferably, the default values for all BLAST 2 search parameters are used, including reward for match=1, penalty for mismatch=-2, open gap penalty=5, extension gap penalty=2, gap x_dropoff=50, expect=10, wordsize=11, and optionally, filter on. In the comparison of two nucleotide sequences using the BLAST search algorithm, structural similarity is referred to as "identities." Preferably, a polynucleotide includes a nucleotide sequence having at least 80% nucleotide identity, at least 81% nucleotide identity, at least 82% nucleotide identity, at least 83% nucleotide identity, at least 84% nucleotide identity, at least 85% nucleotide identity, at least 86% nucleotide identity, at least 87% nucleotide identity, at least 88% nucleotide identity, at least 89% nucleotide identity, at least 90% nucleotide identity, at least 91% nucleotide identity, at least 92% nucleotide identity, at least 93% nucleotide identity, at least 94% nucleotide identity, at least 95% nucleotide identity, at least 96% nucleotide identity, at least 97% nucleotide identity, at least 98% nucleotide identity, or at least 99% nucleotide identity to SEQ ID NO:17. Preferably, a nucleotide sequence having structural similarity to SEQ ID NO:17 encodes a compound of the present invention having biological activity.

[0064]Optionally, a polynucleotide identical to, or having structural similarity with SEQ ID NO:17 includes an additional nucleotide sequence located immediately 5' or upstream of SEQ ID NO:17. This optional sequence encodes a polypeptide corresponding to the amino terminal region of the prepeptide that is removed during processing, i.e., amino acids 1-33 of SEQ ID NO:6. These nucleotides are disclosed at nucleotides 1-99 of SEQ ID NO:5. It should be understood that a polynucleotide encoding a polypeptide represented by amino acids 1-33 of SEQ ID NO:6 is not limited to the nucleotide sequence disclosed at nucleotides 1-99 of SEQ ID NO:5, but also includes the class of polynucleotides encoding such a polypeptide as a result of the degeneracy of the genetic code.

[0065]Other isolated polynucleotides encoding the amino terminal region of the prepeptide include those having structural similarity with the nucleotide sequence of nucleotides 1-99 of SEQ ID NO:5. The similarity is referred to as structural similarity and is determined by aligning the residues of the two polynucleotides (i.e., the nucleotide sequence of the candidate sequence and nucleotides 1-99 of SEQ ID NO:5) as described above. Preferably, such a polynucleotide includes a nucleotide sequence having at least 80% nucleotide identity, at least 81% nucleotide identity, at least 82% nucleotide identity, at least 83% nucleotide identity, at least 84% nucleotide identity, at least 85% nucleotide identity, at least 86% nucleotide identity, at least 87% nucleotide identity, at least 88% nucleotide identity, at least 89% nucleotide identity, at least 90% nucleotide identity, at least 91% nucleotide identity, at least 92% nucleotide identity, at least 93% nucleotide identity, at least 94% nucleotide identity, at least 95% nucleotide identity, at least 96% nucleotide identity, at least 97% nucleotide identity, at least 98% nucleotide identity, or at least 99% nucleotide identity to nucleotides 1-99 of SEQ ID NO:5.

[0066]The present invention also includes isolated polynucleotides encoding the 7 polypeptides that facilitate production of a compound of the present invention. These polynucleotides include SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:15. These polynucleotides encode SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16, respectively. It should be understood that a polynucleotide encoding a polypeptide represented by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 is not limited to the nucleotide sequence disclosed at SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15, but also includes the class of polynucleotides encoding such a polypeptide as a result of the degeneracy of the genetic code.

[0067]Other polynucleotides encoding on of the 7 polypeptides that facilitate expression of a compound of the present invention include those having structural similarity with the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15. The similarity is referred to as structural similarity and is determined by aligning the residues of the two polynucleotides (e.g., the nucleotide sequence of the candidate sequence and the nucleotide sequence of SEQ ID NO:1) as described above. Preferably, a polynucleotide includes a nucleotide sequence having at least 80% nucleotide identity, at least 81% nucleotide identity, at least 82% nucleotide identity, at least 83% nucleotide identity, at least 84% nucleotide identity, at least 85% nucleotide identity, at least 86% nucleotide identity, at least 87% nucleotide identity, at least 88% nucleotide identity, at least 89% nucleotide identity, at least 90% nucleotide identity, at least 91% nucleotide identity, at least 92% nucleotide identity, at least 93% nucleotide identity, at least 94% nucleotide identity, at least 95% nucleotide identity, at least 96% nucleotide identity, at least 97% nucleotide identity, at least 98% nucleotide identity, or at least 99% nucleotide identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15. Preferably, a nucleotide sequence having structural similarity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15 having the activity of producing a compound of SEQ ID NO:22. Testing for such activity is described above.

[0068]A polynucleotide of the present invention may be present in a vector. A vector is a replicating polynucleotide, such as a plasmid, phage, cosmid, or artificial chromosome, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. When present in a vector, a polynucleotide of the invention may be referred to as a recombinant polynucleotide. As used herein, a "recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally joined together. The sequences may be joined by the artificial manipulation of different polynucleotide sequences using recombinant techniques, or may be chemically or enzymatically synthesized. A recombinant polynucleotide may be included in a suitable vector. Construction of vectors containing a polynucleotide of the invention employs standard ligation techniques known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989).

[0069]A vector can provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polypeptide encoded by the coding region, i.e., an expression vector. Suitable expression vectors include those that can be used to produce amounts of polypeptide, preferably a compound of the present invention that can be used in a composition of the present invention and, for instance, administered to a subject. Vectors may include a coding region encoding a polypeptide of the present invention or a fragment thereof. As used herein, a "coding region" refers to a nucleotide sequence that encodes a polypeptide and, when placed under the control of appropriate regulatory sequences expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end

[0070]Selection of a vector depends upon a variety of desired characteristics in the resulting construct, such as a selection marker, vector replication rate, and the like. Suitable host cells for cloning or expressing the vectors herein are prokaryote or eukaryotic cells.

[0071]An expression vector optionally includes regulatory sequences operably linked to the coding region. A regulatory sequence is a nucleotide sequence that regulates expression of a coding region to which it is operably linked. Nonlimiting examples of regulatory sequences include promoters, transcription initiation sites, translation start sites, translation stop sites, and terminators. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A regulatory sequence is "operably linked" to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence. The invention is not limited by the use of any particular promoter, and a wide variety of promoters are known. Promoters act as regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3' direction) coding region. The promoter used may be a constitutive or an inducible promoter. It may be, but need not be, heterologous with respect to the host cell. As used herein, a "heterologous" regulatory sequence is a regulatory sequence operably linked to a coding region to which it is not normally operably linked.

[0072]An expression vector may optionally include a ribosome binding site and a start site (e.g., the codon ATG) to initiate translation of the transcribed message to produce the polypeptide. It may also include a termination sequence to end translation. A termination sequence is typically a codon for which there exists no corresponding aminoacetyl-tRNA, thus ending polypeptide synthesis. The polynucleotide used to transform the host cell may optionally further include a transcription termination sequence.

[0073]A vector may include more than one polynucleotide of the present invention. When more than one polynucleotide of the present invention is present in one vector, the polynucleotides may be organized in an operon, and operably linked to the same promoter located upstream of the first coding region in the operon. Alternatively, more than one promoter may drive expression of the polynucleotides. For instance,

[0074]The vector introduced into a host cell optionally includes one or more marker sequences, which typically encode a molecule that inactivates or otherwise detects or is detected by a compound in the growth medium. For example, the inclusion of a marker sequence may render the transformed cell resistant to an antibiotic, or it may confer compound-specific metabolism on the transformed cell. Examples of a marker sequence are sequences that confer resistance to kanamycin, ampicillin, chloramphenicol, tetracycline, neomycin, and eruthromycin.

Methods of Making

[0075]The present invention is also directed to methods for making compounds of the present invention. A method for producing a compound of the present invention may include growing a Bifidobacterium under conditions suitable for producing the compound. Typically, such conditions may include growing the Bifidobacterium on a surface. Suitable components that cam be used to for a solid medium include, but are not limited to, agar, gelatin, and gums such as alginate, xantham, and the like. The medium may be complete or minimal, preferably complete. Examples of suitable media include, but are not limited to, complex media that include a fermentable sugar, such as MRS, BLIM, and Brain Heart Infusion.

[0076]Bifidobacteria that may produce a compound of the present invention can be obtained from an individual, or laboratory strains can be used. Examples of bifidobacteria that may be used as a source of a compound of the present invention include B. adolescentis, B. aerophilum, B. angulatum, B. animalis, B. asteroides, B. bifidum, B. boum, B. breve, B. catenulatum, B. choerinum, B. coryneforme, B. cuniculi, B. denticolens, B. dentium, B. gallicum, B. gallinarum, B. indicum, B. infantis, B. inopinatum, B. longum, B. magnum, B. merycicum, B. minimum, B. pseudocatenulatum, B. pseudolongum, B. psychraerophilum, B. pullorum, B. ruminantium, B. saeculare, B. scardovii, B. subtile, B. thermacidophilum, and B. thermophilum. Preferably, the Bifidobacerium is B. breve, B. infantis, or B. longum, more preferably, B. longum.

[0077]Since bifidobacteria are believed to lose the ability to produce lantibiotics after prolonged in vitro culture in liquid medium, a Bifidobacterium is preferably obtained from an individual. Methods for obtaining a Bifidobacterium from an individual are routine and known in the art (see, for instance, Kullen et al., 1997, FEMS Microbiol. Lett., 154:377-383; O'Suillivan, U.S. Pat. No. 6,746,672). For instance, fresh fecal samples may be collected from an individual and immediately homogenized in an appropriate amount of a sterile solution such as sterile peptone water (0.1%). Preferably, an individual has no history of gastrointestinal disorders and has not used antibiotics in the previous year. The homogenate may be transferred to an anaerobic chamber, where it may be serially diluted and plated on, for instance, BIM-25 (Munoa et al., 1988, Appl. Environ. Microbiol., 54:1715-1718). After anaerobic incubation at 37° C., red colonies can be randomly selected. The authenticity of the colonies appearing on the BIM-25 plates can be verified by routine methods, such as assessing the activity of fructose-6-phosphate phosphoketolase, a diagnostic enzyme for bifidobacteria, or by molecular analysis of the 16s rRNA gene or the recA gene as described by Kullen et al. (1997, FEMS Microbiol Lett., 154:377-383).

[0078]Once a microbe such as a Bifidobacterium is growing in conditions that permit lantibiotic production it is expected to be able to continue to do so. However, a Bifidobacterium that produces a compound of the present invention should not be grown for extended periods under conditions that do not favor lantibiotic production, such as in broth, as they can loose the gene cluster encoding the compound. As the gene cluster also encodes the immunity genes, it cannot be lost if the lantibiotic is in the environment.

[0079]A Bifidobacterium can be screened to determine if it produces a compound of the present invention. Screening methods include culturing a Bifidobacterium under conditions suitable for expression of a lantibiotic and testing for the presence of a lantibiotic. Conditions that are "suitable" for an event to occur, or conditions that "allow" an event to occur, such as production of a lantibiotic by a Bifidobacterium, or "suitable" conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event. Methods for determining whether a Bifidobacterium expresses a compound of the present invention are described above.

[0080]Screening methods may include determining if a Bifidobacterium has one or more of the polynucleotides involved in the synthesis of a compound of the present invention. For instance, the presence of a polynucleotide of the present invention can be determined by amplification. Preferably, a polynucleotide is amplified by the polymerase chain reaction (PCR). In PCR, a molar excess of a primer pair is added to a sample that includes polynucleotides from the test Bifidobacterium, preferably the chromosomal DNA. The primers are extended to form complementary primer extension products which act as templates for synthesizing the desired amplified polynucleotides. The presence of an amplified polynucleotide of the expected size indicates the test Bifidobacterium may produce a compound of the present invention.

[0081]Suitable polynucleotides that can be amplified include coding regions present in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15. Preferably, the polynucleotide amplified is a portion of the nucleotide sequence (SEQ ID NO:5) encoding the polypeptide SEQ ID NO:6. Primers that amplify a portion of a polynucleotide of the present invention can be designed using readily available computer programs, such as OMIGA program, (Oxford Molecular, Ltd., Oxford, UK). Factors that can be considered in designing primers include, but are not limited to, melting temperatures, primer length, size of the amplification product, and specificity. Primer length is generally between 15 and 30 nucleotides, but can be shorter or longer if desired. The conditions for amplifying a polynucleotide by PCR vary depending on the nucleotide sequence of primers used, and methods for determining such conditions are routine in the art. Examples of primer pairs include, for instance, LANR1-F (ATGAAGGCGATTCTGTTTC, SEQ ID NO:38) and LANR1-R (TCACAGCTCGATATTGGTG, SEQ ID NO:39), which result in an amplified product of 676 bp, and LANT1-F (GAGCATCAATGAGAAGTCC, SEQ ID NO:56) and LANT1-R (GCAATCAACACCAAAACC, SEQ ID NO:57), which result in an amplified product of 788 bp.

[0082]In another aspect the presence of a polynucleotide of the present invention can be determined with polynucleotide probes designed to hybridize to a polynucleotide present in the test Bifidobacterium. As used herein, "hybridizes," "hybridizing," and "hybridization" refers to noncovalent interaction forms between a probe and a target polynucleotide under standard conditions. Standard hybridizing conditions are those conditions that allow a probe to hybridize to a target polynucleotide. Such conditions are readily determined for a probe and the target polynucleotide using techniques well known to the art, for example see Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory: New York (1989). Suitable polynucleotides that can be identified by hybridization include coding regions present in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15. Preferably, the polynucleotide identified by hybridization is the nucleotide sequence encoding the polypeptide SEQ ID NO:6. A probe may be less than 20 nucleotides, at least 20 nucleotides, at least 50 nucleotides, or at least 100 nucleotides in length.

[0083]In another aspect, a method for producing a compound of the present invention may include growing a microbe that includes a recombinant polynucleotide encoding a polypeptide that includes an amino acid sequence having structural similarity to SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22, preferably SEQ ID NO:22. A microbe may include a coding region encoding a lanA of the present invention, and optionally may include a coding region encoding a lanR2 of the present invention, a lanK of the present invention, a lanR1 of the present invention, a lanD of the present invention, a lanM of the present invention, a lanI of the present invention, a lanT of the present invention, or a combination thereof. Preferably, microbe may include a coding region encoding SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or a combination thereof. A compound of the present invention may also be produced in vivo (Xie et al., 2004, Science, 303:679-681).

[0084]A microbe that includes a recombinant polynucleotide encoding a compound of the present invention may be an Archae, Eukarya or a Eubacteria, preferably a Eubacteria, such as a gram negative or a gram positive microbe. Examples of gram negative microbes include, but are not limited to, E. coli and Salmonella spp. Examples of gram positive microbes include, but are not limited to, Bacillus spp. such as B. subtilis, Enterococcus spp. such as E. faecium, E. faecalis, lactic acid bacteria such as Lactococcus lactis, L. sakei, and Streptomyces. Other microbes include yeast such as, but not limited to, Saccharomyces cerevisiae and Pichia pastoris.

[0085]A compound of the present invention may be isolated. For instance, a microbe producing a compound of the present invention, preferably a Bifidobacterium, can be grown in conditions suitable for the production of a lantibiotic, and the culture, including the medium, exposed to conditions suitable for isolating the compound. In one aspect, a compound of the present invention may be isolated by drying the cells and, optionally, the solid medium on which the cells are grown. Optionally, the culture may be further treated to sterilize it. For example, the culture can be treated by exposure to conditions to kill the bifidobacteria present in the culture. Examples of conditions useful for sterilization include heat or ultraviolet radiation. The culture may be dried until essentially all moisture is removed and a powder containing the compound remains. Methods for drying cultures are known to the art and include, for instance, spray drying, freeze drying, tunnel drying, vacuum drying, and air drying. The result of such methods is a mixture that includes a large number of components, including the compound of the present invention. Such a mixture may be added to food products. The mixture added to food products may be sterile.

[0086]In another aspect, a lantibiotic of the present invention may be isolated by methanol extraction. Additional methods may be used for further isolation and/or purification using methods known in the art for isolating and/or purifying lantibiotics. Such methods typically include, but are not limited to, column chromatography, including hydrophobic interaction chromatography, and high performance liquid chromatography (HPLC), such as reverse phase HPLC using, for instance, a C18 column. The optimum conditions to be used can be determined by routine experimentation. A purified compound of the present invention may be made using known synthetic chemistry techniques.

Compositions

[0087]The present invention also provides compositions. A composition may include a compound of the present invention. Such compositions may optionally include a pharmaceutically acceptable carrier. As used herein "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration and not deleterious to a recipient thereof. The compound present in the composition may be isolated or purified. An isolated compound may be one that is isolated by drying the cells. Additional active compounds can also be incorporated into the compositions.

[0088]Compositions of the present invention may further include at least one component that damages the outer membrane of a gram negative microbe. For instance, a composition may include at least one chelator, preferably a metal chelator. The use of chelators such as ethylenediaminetetraacetic acid (EDTA) with lantibiotics is known to result in expanding the activity of some lantibiotics, such as nisin, from just gram positive microbes to include gram negative microbes (Blackburn et al., U.S. Pat. No. 5,691,301). The use of a chelator with a compound of the present invention is not required for the compound to be active against gram negative microbes. Examples of metal chelators include natural and synthetic compounds. Examples of natural compounds include plant phenolic compounds, such as flavonoids. Examples of flavinoids include the copper chelators catechin and naringenin, and the iron chelators myricetin and quercetin. Examples of synthetic copper chelators include, for instance, tetrathiomolybdate, and examples of synthetic zinc chelators include, for instance, N,N,N',N'-Tetrakis(2-pyridylmethyl)-ethylene diamine. Examples of synthetic iron chelators include 2,2'-dipyridyl, 8-hydroxyquinoline, EDTA, ethylenediamine-di-O-hydroxyphenylacetic acid (EDDHA), desferrioxamine methanesulphonate (desferol), transferrin, lactoferrin, ovotransferrin, biological siderophores, such as xcatecholates and hydroxamates, and citric acid. Preferably, the chelator is EDTA.

[0089]Compositions of the present invention may further include at least one surfactant, preferably a non-ionic surfactant. Examples of non-ionic surfactants include glycerol monolaurate, sucrose esters such as sucrose palmitate, polysorbate 20, TRITON X-100, Isoceteth-20, ARLASOLVE 200L, Lauramine oxide, Decylpolyglucose, Phospholipid PTC, MEROXAPOL 105, and the like.

[0090]Compositions of the present invention may include other agents having bacteriostatic and/or bacteriocidal activity. Examples include, but are not limited to, lysostaphin, bacitracin, neomycin, polymixin, beta-lactams, including penicillin, methicillin, moxalactam and cephalosporins, such as cefaclor, cefadroxil, cefamandole nafate, cefazolin, cefixime, cefinetazole, cefonioid, cefoperazone, ceforanide, cefotanme, cefotaxime, cefotetan, cefoxitin, cefpodoxime proxetil, ceftazidime, ceftizoxime, ceftriaxone, cefriaxone, cefuroxime, cephalexin, cephalosporin C, cepahlosporin C sodium salt, cephalothin, cephalothin sodium salt, cephalothin dihydrate, cephapirin, cephradine, cefuroximeaxetil, loracarbef, and the like, glycopeptides, anti-bacterial enzymes, including anti-staphylococcal enzymes such as mutanolysin, lysozyme or cellozyl muramidase, anti-bacterial antibodies, other anti-bacterial peptides such as defensins, and bacteriocins, including other lantibiotics such as nisin, subtilin, epidermin, cinnamycin, duramycin, ancovenin and Pep 5. In some aspects these agents may be particularly preferred when the composition is to be applied topically.

[0091]A composition may contain organic acids acceptable for use in food products or salts of these acids. A composition may contain individual acids or salts, or mixtures thereof. Preferred organic acids or salts for use in compositions include acetic acid, sodium acetate, sodium diacetate, potassium acetate, lactic acid, sodium lactate, potassium lactate, propionic acid, propionates, including, but not limited to, sodium propionate and potassium propionate, citric acid or its salts such as sodium citrate or potassium citrate, or combinations thereof.

[0092]A composition for administration to a subject may be prepared by methods known in the art of pharmacy. In general, a composition can be formulated in a dosage form to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, perfusion; parenteral, e.g., intravenous, intradermal, intramuscular, subcutaneous; topical, e.g., mucosal (such as nasal, sublingual, vaginal, buccal, or rectal) and transdermal; otic; and oral. Solutions or suspensions can include the following components: a sterile diluent such as water for administration, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; buffers such as acetates, citrates or phosphates; electrolytes, such as sodium ion, chloride ion, potassium ion, calcium ion, and magnesium ion, and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. A composition can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0093]Compositions can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). A composition is typically sterile and, when suitable for injectable use, should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and optionally preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Prevention of the action of microorganisms can be achieved by various optional antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0094]Sterile solutions can be prepared by incorporating the active compound (i.e., a compound of the present invention) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0095]Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a dentifrice. A dentifrice may be a liquid, paste, or powder, such as a mouthwash or a toothpaste. Pharmaceutically compatible binding agents may be included as part of the composition. The tablets, pills, capsules, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. In another aspect, a composition may be a transgenic plant expressing a compound of the present invention.

[0096]For administration by inhalation, the active compounds may be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as a hydrofluoroalkane, or a nebulizer.

[0097]For topical administration compositions of the invention may include various mixtures and combinations that can be applied topically and to permit even spreading and absorption into cutaneous and mucosal surfaces. Examples include sprays, mists, aerosols, lotions, creams, aqueous and non-aqueous solutions or liquids, oils, gels, powders, ointments, pastes, unguents, emulsions and suspensions. Topical formulations may be prepared by combining a compound of the present invention with conventional pharmaceutical or cosmeceutical diluents or carriers commonly used in topical dry, liquid, cream and aerosol formulations. Both liquids and powders can be delivered as sprays, mists or aerosols.

[0098]Powders may be formed with the aid of any suitable powder base, e.g., talc, lactose, starch, and the like. Solutions can be formulated with an aqueous or non-aqueous base, and can also include one or more dispersing agents, suspending agents, solubilizing agents, and the like. Topical gels may be prepared using polymers having a molecular weight and level of concentration effective to form a viscous solution or colloidal gel of an aqueous or non-aqueous solution or suspension of the active compound. Polymers from which topical gels may be prepared include polyphosphoesters, polyethylene glycols, high molecular weight poly(lactic) acids, hydroxypropyl celluloses, chitosan, polystyrene sulfonates, and the like.

[0099]Ointments, creams and lotions may be formulated, for example, with an aqueous or oily base and addition of a suitable thickening agent, gelling agent, stabilizing agent, emulsifying agent, dispersing agent, suspending agent, or consistency regulating agent, and the like. Bases include water, an alcohol or an oil, such as liquid paraffin, mineral oil, or a vegetable oil, such as peanut or castor oil. Thickening agents that can be used according to the nature of the base include soft paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol, polyethylene glycols, polyphosphoesters, poly(lactic acids), hydroxyethyl celluloses, hydroxypropyl celluloses, cellulose gums, acrylate polymers, hydrophilic gelling agents, chitosan, polystyrene sulfonate, petrolatum, woolfat, hydrogenated lanolin, beeswax, and the like.

[0100]The ointments, pastes, creams, gels, and lotions can also contain excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, zinc oxide, and mixtures thereof. Powders and sprays can also contain excipients such as silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Solutions, suspensions or dispersions can be converted into aerosols or sprays by any of the known means routinely used for making aerosols for topical application. In general, such methods include pressurizing or providing a means of pressurizing a container of a solution, suspension or dispersion, usually with an inert carrier gas, and passing the pressured gas through a small orifice. Sprays and aerosols can also contain customary propellants, e.g., chlorofluorohydrocarbons or volatile unsubstituted hydrocarbons, such as butane and propane.

[0101]Excipients may include compounds that promote skin absorption, such as dimethyl sulfoxide (DMSO), partial glycerides of fatty acids, and the like. Examples of partial fatty acid glycerides include, but are not limited to IMWITOR 742 and IMWITOR 308. The topical formulations may also optionally include inactive ingredients to improve cosmetic acceptability, including but not limited to, humectants, surfactants, fragrances, coloring agents, emollients, fillers, and the like.

[0102]A composition may be administered directly by the dusting of a powder, spraying of an aerosol or by spreading a film of an ointment, cream, lotion, solution or gel to the desired area of the skin using the fingertips of the patient or a healthcare provider or other conventional application such as a swab or wipe. The product may be first applied to the skin and spread with the fingertips or an applicator or applied to the fingertips and spread over the skin. The compositions may also optionally first be coated on the surface of a topical applicator, such as a bandage, swab, moist woven or non-woven wipe and the like, which is then applied to the portion of the skin to receive the composition.

[0103]The active compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0104]The active compounds may be prepared with carriers that will protect the active compound against rapid elimination from the body, such as a controlled release formulation, including implants. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. The materials can also be obtained commercially. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0105]Since the lantibiotic described in the examples is expressed by bifidobacteria while present in animals, it is expected the compounds of the present invention are safe and suitable for use in animals, including use in foods eaten by an animal. However, toxicity and therapeutic efficacy of such active compounds may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit high therapeutic indices are preferred. Methods for evaluating the toxicity of lantibiotics are known in the art and are routine.

[0106]The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in animals, including humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For a compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of signs) as determined in cell culture. Such information can be used to more accurately determine useful doses.

[0107]In those aspects where a composition is being administered to an animal for a pharmaceutical application or a personal care application, the composition can be administered one or more times per day to one or more times per week, including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with an effective amount of a compound of the present invention can include a single treatment or, preferably, can include a series of treatments.

[0108]The present invention includes both patient-specific dosages forms, as well as non-patient-specific multi-dosage forms that can be used to decontaminate populations exposed to pathogens as a consequence of a bioterrorism attack.

[0109]A composition of the present invention may include a microbe, such as a Bifidobacterium, that expresses a compound of the present invention. A composition including a microbe that expresses a compound of the present invention may be encapsulated in, for example, a sugar matrix, a fat matrix, a polysaccharide matrix, or a protein matrix. It may also be coated and/or incorporated into tablet form. For instance, encapsulation, coating, and incorporation into tablet form may allow better survival of the microbe in the composition, or may allow better delivery of the microbe to the large intestine.

[0110]Such compositions that include a microbe that expresses a compound of the present invention are often orally administered to an animal. It is known in the art that bifidobacteria can be incorporated into different types of food products. In particular, the bifidobacteria of the present invention can be incorporated into solid and semi-solid dairy products, including fermented dairy products, for instance yogurt. Other examples of dairy products include cottage cheese, cheese, and powdered milk. Bifidobacteria can also be incorporated into baby foods. Beverages to which bifidobacteria can be added include milk, vegetable juice, fruit juice, soy milk, soybean milk, fermented soybean milk, and fruit flavored dairy beverages.

Methods of Use

[0111]The present invention is also directed to methods of using the compositions described herein. The methods include, for instance, pharmaceutical applications, food applications, personal care applications, and probiotic applications. The methods may include preventing microbial growth. The prevention of growth may be due to a bacteriostatic activity or a bacteriocidal activity of a compound of the present invention. The microbe may be gram positive or gram negative. Examples of gram positive microbes that may be sensitive to a lantibiotic of the present invention and inhibited include, but are not limited to, Streptococcus spp., such as S. agalactiae; Enterococcus spp., such as E. faecalis and E. faecium; Bacillus spp., such as B. anthracis, B. cereus, B. coagulans, and B. licheniformis; Listeria spp., such as L. monocytogenes; Staphylococcus spp., such as S. aureus; Streptococcus spp., such as S. agalactiae, S. mutans, S. viridans, S. thermophilus, S. constellatus, and S. zooepidemicus; Clostridium spp., such as C. botulinum, C. difficile, C. perfringens, C. sordellii, C. tetani, C. sordellii, C. sporogenes, C. tyrobutyricum, and C. putrefasciens; Actinomyces spp., such as A. israelii and A. naeslundii; Leuconostoc spp.; Lactobacillus spp.; Micrococcus spp., Mycobacterium spp., Corynebacterium spp., Propionibacterium spp., Pediococcus spp., Peptostreptococcus spp., Sporolactobacillus spp., Brevibacterium spp., and Sporolactobacillus spp.

[0112]Examples of gram negative microbes that may be sensitive to a lantibiotic of the present invention and inhibited include, but are not limited to, members of the family Enterobacteriaceae, such as Citrobacter spp., Edwardsiella spp., Enterobacter spp., Erwinia spp., Escherichia, such as E. coli (e.g., H7:O157), Ewingella spp., Klebsiella, such as K. pneumoniae spp., Plesiomonas, such as P. shigelloides spp., Proteus, such as P. vulgaris spp., Providencia spp., Salmonella spp., Serratia, such as S. marcescens spp., Shigella spp., and Yersinia, such as Y. enterocolitica and Y. pestis; members of the family Vibrionaceae, such as Vibrio alginolyticus, V. cholerae, V. parahaemolyticus, and V. vulnificus; and members of the family Pseudomonadaceae, such as Pseudomonas aeruginosa, P. anguilliseptica, P. oryzihabitans, P. plecoglossicida, P. fluorescens and P. syringae. Other examples of gram negative microbes that may be inhibited include, but are not limited to, Helicobacter pylori; Camplyobacter spp., such as C. jejuni, C. coli, and C. upsaliensis; Bacteroides spp., such as B. frgilis; Fusobacaterium spp., such as F. necrophorum, F. ulcercans, F. russi, and F. varium; Leptospira spp.; Pectobacterium spp., such as P. carotovorum; Pasteurella spp., such as P. multocida, Borrelia spp., Legionella spp., Neissaria spp., Fusobacterium spp., and Agrobacterium spp.

[0113]Pharmaceutical and personal care applications include, for instance, methods of treating an animal to inhibit, preferably prevent microbial growth. As used herein, "treatment" and "treating" refer to the use of a composition of the present invention to prevent, cure, retard, or reduce the severity of signs in a subject resulting from the presence of a microbe, and/or result in no worsening of signs over a specified period of time in an subject which has already been exposed to a microbe that can cause the signs. Treatment may be prophylactic or, alternatively, may be initiated after the exposure of an animal to a microbe. Prophylactic treatment refers to the use of a composition of the present invention to inhibit, preferably prevent microbial growth, thereby preventing or reducing signs of a condition if the subject is later exposed to a microbe. Treatment that is prophylactic, for instance, initiated before a subject manifests signs of a condition, is referred to herein as treatment of a subject that is "at risk" of developing a condition. Treatment initiated after the exposure of a subject to a microbe causing a condition may result in decreasing the severity of the signs, or completely removing the signs.

[0114]As used herein, the term "signs" refers to objective evidence in a subject of a condition caused by the presence of a microbe. Signs can vary depending upon the microbe. Signs of conditions caused by the presence of a microbe and the evaluation of such signs are routine and known in the art. Accordingly, the present invention is also directed to methods for treating a microbial infection in an animal, and methods for treating a condition caused by a microbe. As used herein, a "microbial infection" refers to a detrimental colonization of an animal by a microbe.

[0115]The methods include administering an effective amount of the composition of the present invention to an animal having an infection and/or signs of a condition caused by a microbe, and determining whether the infection and/or signs of the condition have decreased. Conditions include, but are not limited to, wound infections, halitosis, caries, systemic infections, and skin infections.

[0116]The methods may include administering a composition of the present invention to an animal. The animal may be any animal susceptible to a condition caused by a microbe including, but not limited to, a vertebrate, more preferably a mammal, or an avian. Examples of mammals include, but are not limited to, a human; a member of the subfamily Bovinae, such as cattle and bison; a member of the subfamily Caprinae, such as sheep and goats; a member of the genus Sus, such as pigs and hogs; companion animals, such as cats and dogs; and a member of the genus Equus, such as horses and donkeys. Examples of birds include, but are not limited to, domesticated birds such as turkeys, chickens, ducks, and geese. Another example of a vertebrate is a fish. A composition of the present invention may be delivered to an animal by methods described herein and known in the art, thereby providing an effective amount to the animal. In this aspect of the invention, an "effective amount" is an amount effective to inhibit growth of a microbe, prevent the manifestation of signs of the condition, decrease the severity of the signs of the condition, and/or complete remove the signs. It is not required that any composition of the present invention completely inhibit growth of all microbes, or completely cure or eliminate all signs of a condition being treated.

[0117]Food applications include, for instance, food preservation by inhibiting microbes that spoil food. The term "food" or "food product" encompasses all edible nutritive substances and compositions, including those intended for human consumption as well as consumption by, for instance, livestock. "Food" and "food product" includes unprocessed, as well as processed, e.g., cooked, nutritive substances and compositions, such as beverages. The expression "present in food" refers to portions of a food that may be resident to harmful bacteria, such as external surfaces, interior surfaces, or the combination thereof.

[0118]A composition of the present invention may be used in connection with a food product that is susceptible to bacterial growth or degradation. These include, but are not limited to, dairy foods, fruits and vegetables, fruit and vegetable derived products, grains and grain derived products, meats, poultry, and seafood. Examples of dairy foods include, but are not limited to, cheese, milk, cream, and fermented dairy foods such as yogurt. Examples of meats include, for instance, ham, beef, salami, chicken, and turkey, including whole parts or processed meat products made therefrom. Other food products include processed food products including ready to eat meals, entrees, and meats, deli salads, mayonnaise, dressings (including salad dressings), sauces and condiments, pastas, soups, edible oils, fish and fish products, egg products, beverages, aseptically packaged foods, as well as mixtures of the foregoing.

[0119]A composition of the present invention may be used by mixing with and/or applying on a blendable food product, but may be applied to a surface of solid food products by a dip, rinse, or spray, or by application to the interior of such products, e.g. by injection. The composition may be applied as a marinade, breading, seasoning rub, glaze, colorant mixture, and the like, or as an ingredient to be mixed with and incorporated into the food product, In still other aspects, the composition may be indirectly placed into contact with the food surface by applying the composition to food packaging materials, such as a casing or a film, and thereafter applying the packaging to the food surface such that the composition comes into contact with the external food surface. The optimum amount to be used will depend upon the particular food product to be treated and the method used to apply the composition to the food and/or the food surface, but can be determined by routine experimentation.

[0120]Probiotic applications of compositions of the present invention include, for instance, use of a microbe, preferably a Bifidobacterium, expressing a compound of the present invention, as a dietary supplement or as a food ingredient. The uses of bifidobacteria as dietary supplements is known in the art and routine. Typically, a Bifidobacterium expressing a compound of the present invention is administered to an animal in need thereof. The Bifidobacterium can be administered as a biologically pure culture, or as a mixed culture. As used herein, a "mixed" culture is one containing a Bifidobacterium and at least one other microbe, preferably a prokaryotic microbe, more preferably a second Bifidobacterium.

[0121]One method of the present invention provides inhibiting the replication of microbes in the gastrointestinal tract, preferably the large intestine, of an animal by administering to an animal a Bifidobacterium that expresses a compound of the present invention. The method may include measuring the presence in the gastrointestinal tract of the microbe to be inhibited, where a decrease in the presence of the microbe in the animal after administration of the Bifidobacterium indicates inhibition of the replication of the microbe in the gastrointestinal tract of the animal.

[0122]The types of microbes whose replication can be inhibited include those present in the gastrointestinal tract of an animal when the Bifidobacterium is administered, and microbes that are introduced to the gastrointestinal tract after the Bifidobacterium is administered. Thus, a Bifidobacterium expressing a compound of the present invention can also be used in a method for inhibiting the establishment of a microbe in the gastrointestinal tract of an animal.

[0123]Another probiotic application includes methods for establishing a Bifidobacterium flora in an animal. Such a flora is expected to competitively inhibit the ability of other microbes to establish themselves as a flora in the gastrointestinal tract. The method includes administering to an animal a Bifidobacterium that expresses a compound of the present invention. The method also includes measuring the presence in the gastrointestinal tract of the Bifidobacterium over a period of time following the administration. A Bifidobacterium flora is considered to be established in an animal when there is at least about 10⁶ of the Bifidobacterium present per gram of feces. Preferably, the animal is an adolescent or adult human or an infant, including an immature, premature, or mature infant. The present method can be used to establish a Bifidobacterium flora in a healthy human, and in humans that have had their normal intestinal flora modified by, for instance, diarrhea or by drug treatment including antibiotic therapy or chemotherapy.

[0124]The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLES

Example 1

[0125]Bifidobacteria are frequently proposed to be associated with good intestinal health primarily because of their overriding dominance in the feces of breast fed infants. However, clinical feeding studies with exogenous bifidobacteria show they don't remain in the intestine, suggesting they may lose competitive fitness when grown outside the gut.

[0126]To further the understanding of genetic attenuation that may be occurring in bifidobacteria cultures, we obtained the complete genome sequence of an intestinal isolate, Bifidobacterium longum DJO10A that was minimally cultured in the laboratory (less than 20 generations), and compared it to that of a culture collection strain, B. longum NCC2705. This comparison revealed colinear genomes that exhibited high sequence identity, except for the presence of 17 unique DNA regions in strain DJO10A and six in strain NCC2705. While the majority of these unique regions encoded proteins of diverse function, eight from the DJO10A genome and one from NCC2705, encoded gene clusters predicted to be involved in diverse traits pertinent to the human intestinal environment, specifically oligosaccharide and polyol utilization, arsenic resistance and lantibiotic production. Seven of these unique regions were suggested by a base deviation index analysis to have been precisely deleted from strain NCC2705 and this is substantiated by a DNA remnant from within one of the regions still remaining in the genome of NCC2705 at the same locus. This targeted loss of genomic regions was experimentally validated when growth of the intestinal B. longum in the laboratory for 1,000 generations resulted in two large deletions, one in a lantibiotic encoding region, analogous to a predicted deletion event for NCC2705. A simulated fecal growth study showed a significant reduced competitive ability of this deletion strain against Clostridium difficile and E. coli. The deleted region was between two IS30 elements which were experimentally demonstrated to be hyperactive within the genome. The other deleted region bordered a novel class of mobile elements, termed mobile integrase cassettes (MIC) substantiating the likely role of these elements in genome deletion events.

[0127]Deletion of genomic regions, often facilitated by mobile elements, allows bifidobacteria to adapt to fermentation environments in a very rapid manner (2 genome deletions per 1,000 generations) and the concomitant loss of possible competitive abilities in the gut.

Results and Discussion

[0128]Genomic sequencing of a minimally cultured B. longum strain. The power of comparative genomics can provide insights into features that are important for a species to survive and compete in its habitat. The genome sequence of the culture collection strain, B. longum NCC2705 (Schell et al., 2002, Proc Natl Acad Sci USA, 99:14422-14427, is available and the ability to compare this genome with one from a strain that was deliberately minimally cultured in vitro may provide new insights to features that may be important for this prominent species from the human large gut. Newly isolated and minimally cultured B. longum strains were characterized and strain DJO10A was selected based on its prominent ability to bacteriostatically inhibit other bacteria through the production of siderophores (O'Sullivan, U.S. Pat. No. 6,746,672), a characteristic that appeared attenuated in all culture collection and commercial bifidobacteria analyzed. It was therefore selected for genomic sequencing as an isolate that likely had minimal attenuation from its origin in the intestine. The complete genome sequence of this strain was deciphered and consisted of one circular chromosome and two cryptic plasmids, pDOJH10L and pDOJH10S that were described previously (Lee and O'Sullivan, 2006, Appl Environ Microbiol 2006, 72:527-535).

[0129]General characteristics of the B. longum DJO10A genome. The chromosome of B. longum DJO10A contained 2,375,792 bp, with 60.15% G+C content and 1,990 encoded genes containing four rRNA operons, 58 tRNAs, 6 insertion sequence (IS) families as well as one prophage (Table 1). Its genomic characteristics were analogous to strain NCC2705, except it contained an extra tRNA_Ser: GCT encoded on its prophage (Ventura et al, 2005, Appl Environ Microbiol 2005, 71:8692-8705). Codon usage analysis showed that this tRNA is the most frequently used tRNA_Ser in the prophage, while it is not the most used tRNA_Ser for the B. longum DJO10A genome (Table 2), pointing to an evolutionary selective pressure for its presence on the prophage. While both genomes contained tRNA's for every amino acid, the corresponding genes for aminoacyl-tRNA synthetases for both asparagine and glutamine are missing, suggesting a reliance on alternative pathways for translation with these amino acids, similar to many other bacteria (Skouloubris et al. 2003, Proc Natl Acad Sci USA 2003, 100:11297-11302, Min et al., 2002, Proc Natl Acad Sci USA 2002, 99:2678-2683). Both these alternative pathways involve gatABC, which is present in both genomes as well as gltX and aspS involved in the glutamine and asparagine alternative translation pathways respectively, substantiating this proposed translation route. Interestingly, the B. longum genome contains novel mobile integrase cassettes (MIC) consisting of three different contiguous integrases flanked by an inverted repeat and a palindrome structure sandwiched by two IS3-type IS elements (FIG. 1). Analysis of the genome of B. longum NCC2705 revealed three analogous MIC elements, located in a non-linear fashion relative to strain DJO10A indicating these elements are indeed mobile (FIG. 9). Interestingly, analysis of the genome sequences of another Bifidobacterium species, B. adolescentis (GeneBank AP009256), as well as other intestinal bacteria, Bacteroides (AE015928), Lactobacillus (CP000033), and E. coli (U00096), did not reveal MIC elements, suggesting these structures may be unique to a subset of closely related bifidobacteria.

TABLE-US-00001 TABLE 1 Overall characteristics of the genomes of B. longum strains DJO10A and NCC2705. DJO10A NCC2705 Size of chromosome (bp) 2,375,792 2,256,640 Overall G + C % 60.15 60.12 Number of plasmids 2 (10 and 3.6 kb) 1 (3.6 kb) Genes Total genes 1990 1727 Average gene length (bp) 1031 1115 Gene density (genes/kb) 0.838 0.765 Gene coding percentage (%) 86.4 85.3 Gene G + C % 61.13 60.86 Unique Sequences Strain-specific unique genes 269 117 Number of unique regions^a 17 6 Number of genes in unique regions 218 84 Prophage 1 1 Number of genes in prophage region 57 19 RNAs and Repeat Sequences rRNA operons 4 4 tRNAs 58 57 Tandem repeats 22 23 Mobile Elements^b Mobile integrase cassette (MIC) 4 3 IS3 family 13 14 IS21 family 10 7 IS30 family 9 5 IS256 family 4 7 ISL3 family 7 12 IS200/605 family 1 1 ^arefers to unique regions that encode functional or hypothetical genes in DNA regions >3 kb, ^bincludes fragmented elements

TABLE-US-00002 TABLE 2 Comparison of serine codon usage between chromosomal and prophage genes in strain DJO10A. Amino Non-prophage genes Prophage genes acid Codon Number Frequency Number Frequency Serine UCU 2416 0.35 20 0.18 UCC^a 16802 2.45 169 1.53 UCA 2277 0.33 33 0.30 UCG 9031 1.32 97 0.88 AGU 1837 0.27 19 0.17 AGC^b 8769 1.28 324 2.94 ^abold indicates the most frequent codon; ^bAGC is compatible to the extra tRNA_Ser in the prophage genome.

[0130]Organization of the origin and terminus of replication. An oriC and terC were found in identical locations in the genome of strain DJO10A and the updated genome sequence of strain NCC2705 (FIG. 9). These regions are extremely highly conserved in both genomes (>99.9% identity) and consist of three oriC clusters and a terC, which is consistent with the predicted replication regions from other bacterial genomes (Mackiewicz et al., 2004, Nucleic Acids Res 2004, 32:3781-3791). However, the location of the observed oriC region in both genomes is slightly different from the predicted location based on genome asymmetry, a feature that has previously been seen in the Helicobacter pylori 26695 genome (Mackiewicz et al., 2004, Nucleic Acids Res 2004, 32:3781-3791, Zawilak et al., 2001, Nucleic Acids Res 2001, 29:2251-2259). As well as the multiple oriC clusters, there are 7 different types of DnaA boxes, consistent with the majority of sequenced genomes and are proposed to be involved in controlling initiation of chromosome replication (Mackiewicz et al., 2004, Nucleic Acids Res 2004, 32:3781-3791).

[0131]Restriction and modification (R-M) systems. The protective role that R-M systems impart on bacteria has been compared to the immune system of higher organisms (Price and Bickle, 1986, Microbiol Sci 1986, 3:296-299). The presence of these systems in numerous bacteria demonstrates their important role for bacterial survival in nature. Both of the B. longum genomes encode type I and two type II R-M systems that are highly conserved (FIG. 10). They also contain an Mrr system that is predicted to restrict methylated DNA (usually HhaII or PstI methylated DNA) that is 100% conserved between both strains (FIG. 10A). The clustering of Mrr with the type I R-M system is similar to E. coli K12 (GenBank U00096). The low identity (40%) between the HsdS proteins in the two strains likely reflects the independent evolution of this type I R-M system in these strains following their evolutionary divergence, as these systems evolve by changing their specificity components (HsdS) to enable it to recognize different sequences. This is substantiated by the existence of an hsdS gene that was inactivated by an IS256 insertion event and both parts of this disrupted gene exhibit much higher conservation, suggesting the insertion event occurred before their evolutionary divergence (FIG. 10A). Upstream from this locus in strain DJO10A there is another restriction gene, McrA (restricts DNA methylated by HpaII or SssI), that is not present in NCC2705. The conserved type II R-M systems in both strains are isoschizomers of Sau3AI and EcoRII which restrict DNA at very frequently occurring sites (FIGS. 10B and 10C). This, together with the range of restriction systems present, may be a factor in limiting the incursion of foreign DNA into these bacteria thus explaining the very low electroporation frequencies reported for bifidobacteria to date.

[0132]Unique genome regions in the B. longum strains. Alignment of the genome sequence of B. longum DJO10A with that of strain NCC2705 illustrates that they are highly conserved and collinear, except for the mobile IS and MIC elements (FIG. 9). There is also an apparent genome reduction in strain NCC2705, consistent with previous observations for microbes growing in a stable environment without horizontal gene transfer opportunities and redundant genes accumulating mutations before subsequent deletion (Nilsson et al., 2005, Proc Natl Acad Sci USA 2005, 102:12112-12116). There are 248 unique sequences of >10 bp between the two genomes, with the majority of them being short and encoding few if any genes. This high number of unique sequences between the two strains was surprising given that the genomes of a clinical isolate of Mycobacterium tuberculosis and one that was extensively passaged for decades in the laboratory display only 86 of such regions in genomes twice the size (Fleischmann et al., 2002, J Bacteriol 2002, 184:5479-5490). There are 23 larger unique regions that encode functional or hypothetical genes and range in size from 3.0 to 48.6 kb, with 17 of these unique regions present in strain DJO10A encoding 219 predicted genes, and 6 unique regions in NCC2705 encoding 84 genes (FIG. 2A). These unique regions are not clustered around the oriC and terC which have previously been associated with regions of intraspecies variation (Berger et al, 2007, J Bacteriol 2007, 189:1311-1321, Molenaar et al., 2005, J Bacteriol 2005, 187:6119-6127).

[0133]One unique region in each genome corresponds to a prophage. The prophage in strain NCC2705, which is truncated, appears to be a longtime resident of the genome as it does not correspond with a Base Deviation Index (BDI) peak (FIG. 2A), as this analysis predicts recent horizontal gene transfer (HGT) events. This appears to have been replaced in the genome of strain DJO10A with a different prophage, that is complete and inducible (Lee and O'Sullivan, 2006, Appl Environ Microbiol 2006, 72:527-535) and corresponds with a significant BDI peak substantiating this recent HGT event. The other five unique regions in strain NCC2705 contain largely hypothetical genes or genes of diverse functions without any significant gene clusters. However one of these regions (unique region 4') does encode putative xylan degradation genes, which is a function predicted to be important for competition in the large intestine. As this region corresponds to a BDI peak, it suggests it may be a recent acquisition by this strain and its evolution in the large intestine would provide the selective pressure for acquiring this unique region. Of the other 16 unique regions in the strain DJO10A, eight encode significant gene clusters involved in functions predicted to be important for competition in the large intestine, specifically oligosaccharide and polyol utilization, arsenic resistance and lantibiotic production.

[0134]Oligosaccharide and polyol utilization. According to a COG functional classification (Tatusov et al., 2000, Nucleic Acids Res 2000, 28:33-36), the highest number of unique genes in strain DJO10A with a predicted function belongs to the carbohydrate metabolism [G] category (Table 3). Interestingly, most of the unique genes in the carbohydrate metabolism category are involved in oligosaccharide utilization, which is the major carbohydrate source available to microbes in the large intestine. In all there are 11 oligosaccharide utilization gene clusters in strain DJO10A, of which 5 are fully present and 2 are partially present in strain NCC2705 (FIG. 11). It is noteworthy that one of these clusters (Cluster 7 in FIG. 11) contains an ISL3 element in the NCC2705 genome at the precise location of the extra oligosaccharide utilization genes in strain DJO10A (FIG. 3). A BDI analysis suggested that the extra oligosaccharide gene clusters in strain DJO10A were not acquired following evolutionary divergence from strain NCC2705, as neither corresponds with a BDI peak (FIG. 2A). The majority of BDI peaks suggesting recent HGT events were the same in both genomes substantiating this analysis. This would suggest the six unique regions 6, 9, 10, 11, 15 and 17 encoding oligosaccharide utilization genes were likely lost from strain NCC2705 during its adaptation to a fermentation environment. Further evidence for the loss of these unique regions from strain NCC2705 comes from a DNA remnant of 361 bp (98% identity) from the ushA gene within the unique region 1 that was left remaining at this locus in NCC2705 (FIG. 2B).

TABLE-US-00003 TABLE 3 COG categories for all genes in both B. longum genomes. B. longum B. longum Function class Individual function categories DJO10A NCC2705 Information J: Translation, ribosomal structure and biogenesis 133 (2)^a 130 K: Transcription 129 (18) 115 (9) L: DNA replication, recombination, and repair 150 (20) 96 (1) Cellular processes D: Cell division and chromosome partitioning 22 (2) 23 (1) V: Defense mechanisms 48 (5) 48 (3) O: Posttranslational modification, protein turnover 51 (2) 50 (2) M: Cell envelope biogenesis, outer membrane 68 (8) 67 (10) P: Inorganic ion transport and metabolism 56 (2) 54 U: Intracellular trafficking, secretion 16 (1) 14 N: Cell motility 3 4 T: Signal transduction mechanisms 53 (6) 41 (1) Metabolism F: Nucleotide transport and metabolism 70 (2) 65 (1) G: Carbohydrate transport and metabolism 188 (32) 167 (7) E: Amino acid transport and metabolism 171 (6) 153 H: Coenzyme metabolism 44 44 (1) I: Lipid metabolism 41 (1) 36 (1) C: Energy production and conversion 50 (1) 50 (2) Q: Secondary metabolites transport and metabolism 6 4 Poorly characterized R: General function prediction only 167 (20) 161 (15) S: Function unknown 525 (142) 405 (63) Total 1991 (270) 1727 (117) ^arefers to the number of genes in the unique regions of each genome as defined in the text

[0135]Polyols are not digestible by humans and their metabolism is believed to be important for bacterial competition in the human large intestine and their ingestion has been implicated in increased bifidobacteria numbers (Gostner et al., 2006, Br J Nutr, 95:40-50.). While strain NCC2705 does not contain genes involved in polyol metabolism, unique region 13 of strain DJO10A is dedicated to this (FIG. 4), containing genes involved in polyol recognition, transport and dehydration, and there are also some polyol metabolism genes in unique region 11. Given that unique region 13 does coincide with a BDI peak (FIG. 2A), it may represent gene acquisition by strain DJO10A. Interestingly, a similar polyol locus was found in B. adolescentis ATCC 15703 at a similar genome location (FIG. 4).

[0136]Arsenic resistance. Other unique regions in strain DJO10A encode gene clusters predicted to be involved in characteristics that would be important for survival and competition in the human intestine. Two operons encoding ATP-dependent arsenic resistance genes are in unique regions 5 and 7 and may be important for intestinal survival as the human intestine contains low concentrations of arsenic from the diet (Ratnaike 2003, Postgrad Med J 2003, 79:391-396). Many intestinal bacteria such as E. coli, Lactobacillus and Bacteroides contain arsenic resistance genes (FIG. 5A), substantiating the competitive advantage for having this ability in the intestine. As the unique regions, 5 and 7, containing these arsenic resistance genes do not correspond to BDI peaks (FIG. 2A), it suggests they may not be recently acquired by strain DJO10A, but rather lost from strain NCC2705. This theory, that adaptation to a pure-culture fermentation environment can result in loss of arsenic resistance, was further substantiated by the exceptional arsenate resistance of strain DJO10A which was 2,000% greater than a fermentation adapted Bifidobacterium isolate (B. animalis subsp. lactis BB12) and 100% greater than E. coli K12 (FIG. 5B). This would suggest that this phenotype is a competitive advantage to intestinal isolates, but not of significance for pure-culture fermentation environments.

[0137]Lantibiotic production. The production of antimicrobial peptides, or bacteriocins, is an important characteristic for bacterial competition in natural environments. One exceptionally broad spectrum class of bacteriocins is the lantibiotics, which are post-translationally modified to form lanthionine residues and to date none have been isolated from any bifidobacteria. A 10.2 kb gene cluster encoding all the genes indicative of a lantibiotic was detected in the unique region 12 of strain DJO10A (FIG. 6A). It was also noted that this unique region did not correspond to a BDI peak, suggesting a likely loss of this region from strain NCC2705. As lantibiotic production would be very advantageous for microbial competition in the intestine, but of no value to a microbe in pure culture, it provides the selective pressure for the loss of this unique region 12 from strain NCC2705.

[0138]Genome attenuation of B. longum in fermentation environments. Given the large number of unique DNA regions in the genome of strain DJO10A, that are predicted to have been lost from strain NCC2705, it suggests that deletion of DNA regions that are not useful may reflect the rapid adaptation of B. longum to a new and very different environment than exists in the human large gut. This would suggest an elevated mutation frequency. A comparative nucleotide substitution analysis between strains DJO10A and NCC2705 shows the majority of genes are highly conserved (FIG. 12), which is to be expected with two closely related strains. However, analysis of the 52 least conserved genes (listed as `positive selection` in FIG. 12) indicates that of the mutations that can be attributed to one strain or the other (frameshifts, deletions, insertions and stop mutations), 11 are from strain NCC2705 and three from strain DJO10A (Table 4). Further substantiation of an increased mutation frequency in strain NCC2705 comes from comparing genes encoding surface protein homologs between the two strains. A search of the DJO10A genome for LPXTG motifs, which is a signature of one class of cell surface anchoring proteins found four potential proteins and SignalP analysis of these proteins (BLD1468, BLD1511, BLD1637 and BLD1638) confirmed the presence of a signal sequence in each case (FIG. 13 Additional file 10). In addition, BLASTP analysis of these four proteins showed that they are very similar to other known surface proteins containing the LPXTG motif. The NCC2705 showed three of these gene homologs (BLD1468, BLD1637 and BLD1638), and had a predicted protein exhibiting 99% amino acid identity to BLD1511, but was missing the LPXTG motif due to an ISL3 insertion in the 3' end of the gene. This further highlights the rapid evolutionary status of bifidobacteria when they are removed from the human gut into pure-culture fermentation environments.

TABLE-US-00004 TABLE 4 Substitution ratios of the 52 genes in the positive selection category. DJO10A NCC2705 dN:dS Annotation Mutation BLD1991 BL1813 20.0673 Hypothetical protein Frameshift (NCC2705) BLD1477 BL1511 14.3257 Hypothetical protein Frameshift and insertion (NCC2705) PyrH pyrH 12.9880 Uridylate kinase Frameshift (NCC2705) BLD0511 BL0781 12.7529 Hypothetical protein Frameshift (NCC2705) BLD0760 BL1007 10.5700 Predicted glycosyltransferase Frameshift (NCC2705) ruvB ruvB 9.3214 Holliday junction resolvasome, helicase subunit citB BL1402 7.5024 Response regulator Deletion (DJO10A) ppa ppa 6.2668 Inorganic pyrophosphatase Deletion (NCC2705) BLD0382 BL1490 6.1532 Hypothetical protein Frameshift (NCC2705) BLD1282 BL0491 4.2133 Hypothetical protein BLD0801 BL1050 4.0425 Hypothetical protein soj BL1492 3.8930 ATPase involved in chromosome partitioning BLD1365 BL0571 3.7661 Predicted esterase Deletion (NCC2705) ardA BL1465 3.6056 Antirestriction protein cbiO BL0049 3.4507 ABC-type cobalt transport system, ATPase component BLD0038 BL0026 3.3177 Hypothetical protein BLD0376 BL1489 3.2395 Hypothetical protein Deletion (DJO10A) dppD oppD 3.1112 ABC-type dipeptide/oligopeptide transport system BLD0144 BL0126 3.0474 Hypothetical protein BLD1389 BL0595 2.4181 Hypothetical protein BLD0109 BL0091 2.3769 Predicted aminoglycoside phosphotransferase srtA BL0676 2.3202 Sortase (surface protein transpeptidase) Deletion (NCC2705) BLD0716 BL0962 1.9561 Predicted acyltransferase metK metK 1.9085 S-adenosylmethionine synthetase Frameshift (NCC2705) BLD1580 BL1246 1.7926 Hypothetical protein BLD1774 BL1650 1.7408 Hypothetical protein lytE BL1311 1.7363 LysM repeat azlC BL1669 1.6564 Predicted branched-chain amino acid permease glgP glgP 1.5354 Glucan phosphorylase BLD1672 BL1553 1.5276 Flagellar basal body P-ring biosynthesis protein BLD1399 BL0605 1.5179 Hypothetical protein ftsW ftsW 1.4969 Uridylate kinase BLD1753 BL1627 1.4775 Predicted transcriptional regulator nagA nagA 1.3702 N-acetylglucosamine-6-phosphate deacetylase BLD0620 BL0885 1.3620 Predicted acyltransferase Stop mutation (DJO10A) DAP2 BL1649 1.3497 Dipeptidyl aminopeptidase/acylaminoacyl-peptidase BLD1741 BL1614 1.3341 Hypothetical protein topB topB 1.3066 Topoisomerase III Annotation difference^a (NCC2705) topA topA 1.2608 Topoisomerase I BLD0571 BL0837 1.2585 Hypothetical protein BLD0397 BL1498 1.2350 Hypothetical protein wecD BL1151 1.2166 Histone acetyltransferase HPA2 pepC pepC2 1.1349 Aminopeptidase C sdrC BL0094 1.1037 Predicted secreted protein containing a PDZ domain BLD1612 BL1278 1.0894 ABC-type transport system BLD1568 rmlB1 1.0865 dTDP-D-glucose 4,6-dehydratase BLD0548 BL0810 1.0694 Hypothetical protein BLD1455 BL0660 1.0642 Serine/Threonine protein kinase BLD0375 BL1488 1.0557 Hypothetical protein Deletion (NCC2705) BLD1772 BL1648 1.0402 Hypothetical protein BLD1401 BL0607 1.0338 Hypothetical protein BLD1983 BL1815 1.0056 Hypothetical protein ^aAn N-terminal extension in the annotation of TopB in strain NCC2705 does not map to any other TopB sequence.

[0139]IS30 `jumping` in the B. longum genome. The dynamic environment within the B. longum cell in a fermentation environment is further substantiated by the intriguing observation during genome sequencing from different batches of DNA that everything was identical except for the location of some IS30 elements (FIG. 7A). This very rapid movement of an IS element within a cell has not been observed previously. The movement of IS30 within the genome occurred only at specific sites, consistent with its insertion target specificity (Olasz et al., 1998, Mol Microbiol 1998, 28:691-704).

[0140]Adaptation of B. longum DJO10A to a pure-culture environment. To test the hypothesis that the switch from a variable and complex environment like the gut to a relatively stable and simplified, pure-culture one, results in hyper IS30 activity and rapid DNA loss of regions that are no longer beneficial to the new environment, strain DJO10A was grown in a typical laboratory medium without pH control for ˜1,000 generations. Isolated colonies were then screened for seven unique regions encoding functions predicted to be useful for survival in the human gut. One of these regions (no. 12) involved in the lantibiotic production was found to be missing from 40% of the isolates (FIG. 14) substantiating this hypothesis. Analysis of this adapted strain, DJO10A-JH1, shows the deletion extended over the full lantibiotic region very similar to strain NCC2705 (FIG. 6A). It is further noted using Pulsed Field Gel Electrophoresis (PFGE) that the 39.9 kb XbaI band containing this region is missing from strain DJO10A-JH1 (FIG. 6B). The loss of the complete lantibiotic gene cluster from 40% of the culture was intriguing as the cluster also encodes the immunity gene to protect cells from the lantibiotic activity. However, analysis of lantibiotic production by strain DJO10A showed that none occurred during growth in broth media, and a solid surface such as agar, was needed for production (FIG. 6C) similar to streptin production from Streptococcus pyogenes (Wescombe and Tagg, 2003, Appl Environ Microbiol 2003, 69:2737-2747). The loss of the complete lantibiotic gene cluster renders strain DJO10A-JH1 sensitive to this pronase-E sensitive lantibiotic, which is also active against a wide spectrum of bacteria (FIG. 6C). Interestingly, the lantibiotic genome region that was deleted during the adaptation of strain DJO10A to the pure-culture environment was located between two IS30 elements, suggesting its role in genome deletion events.

[0141]It was also noted that the pure-culture adapted strain, DJO10A-JH1, was also missing a 140.7 kb XbaI band (FIG. 6B). It is intriguing that this band contains one of the four MIC elements, suggesting it may have been involved. PCR analysis of the loci immediately bordering this MIC element revealed the deletion extended between 10 and 50 kb directly downstream from this element substantiating its likely role in this deletion event. This further substantiated the rapid loss of DNA, and the prominent role of mobile elements, during evolutionary adaptation by these bacteria.

[0142]Southern hybridization of strains DJO10A and DJO10A-JH1 substantiate the IS30 `jumping` during growth in a pure-culture environment, while the positions of the other IS elements (IS21, IS256 and ISL3) remained stable (FIG. 7B). This IS30 hyperactivity in B. longum genomes may play an important role in deletion events and genome reduction during adaptation to new environments.

[0143]Competitive `fitness` of the adapted B. longum strain DJO1A-JH1. The rapid genome reduction experienced by B. longum DJO10A during pure-culture growth in fermentation conditions suggested that the genomic regions lost may have been important for competition in the intestine. To test if this in vitro adaptation affected the `fitness` of the strain, a simulated fecal competitive approach was developed. Bifidobacteria are frequently proposed to successfully compete against members of the clostridia and the enterobacteriae in the intestinal environment. A member of both of these bacterial groups was selected to test the relative competitive abilities of B. longum DJO10A and its in vitro adapted derivative, strain DJO10A-JH1. To ensure that the selected competitor strains were not attenuated in any way, new isolates were obtained from fresh feces by plating on selective media and speciated using a sequence analysis of the 16S rRNA gene. This resulted in the isolation of Clostridium difficile DJOcd1 and E. coli DJOec1, which were minimally cultured prior to undergoing fecal competitive experiments with the B. longum strains. An in vitro growth rate analysis established that E. coli DJOec1 had the fastest growth rate, followed by C. difficile DJOcd1, B. longum DJO10A-JH1 and B. longum DJO10A (FIG. 15). The noticeable increased growth rate of B. longum DJO10A-JH1 compared to strain DJO10A substantiated that the genome reduction of strain DJO10A-JH1 favored the in vitro growth environment.

[0144]Competitive growth experiments with both E. coli DJOec1 and C. difficile DJOcd1 in a simulated anaerobic fecal environment revealed that B. longum DJO10A had a significantly greater ability to compete against both E. coli and C. difficile (FIG. 8). The significantly greater reduction in both these groups of bacteria by B. longum DJO10A supports the genome analysis hypothesis that the genome reduction exhibited in pure-culture growth may compromise a bacterium's ability to compete in its original environment.

[0145]While the simulated fecal competition studies suggested that the lantibiotic encoding genome region was important for competition in the human intestine, in vivo studies in an intestinal model would be necessary to verify this hypothesis.

Methods

[0146]Bacterial strains and growth conditions. Bifidobacterium longum strain DJO10A was isolated from a healthy young adult's feces (Islam, 2006, MS thesis. University of Minnesota, Department of Food Science and Nutrition) and B. animalis subsp. lactis BB12 was obtained from Chr. Hansen (Denmark). B. animalis subsp. lactis strains S1, S2, and S14 are genetically distinct isolates from fermented foods. Clostridium difficile DJOcd1 was isolated from fresh feces by plating on Clostridium difficile Selective Agar (BD Diagnostics) and speciated using a sequence analysis of its 16S rRNA gene. E. coli DJOec1 was obtained from fresh feces by plating on MacConkey agar (Difco) and speciated using a sequence analysis of its 16S rRNA gene. E. coli K12 was obtained from the American Type Culture Collection (ATCC). Bifidobacteria were cultivated at 37° C. in MRS (Difco) supplemented with 0.05% L-cysteine HCl (Sigma), Bifidobacteria Low-Iron Medium (BLIM) (Islam, 2006, MS thesis. University of Minnesota, Department of Food Science and Nutrition) or Bifidobacteria Fermentation Medium (BFM) (2% proteose peptone, 0.15% K2HPO4, 0.15% MgSO4.7H2O, 0.5% D-glucose) under anaerobic conditions using either the BBL Anaerobic system (BBL) or the Bactron II Anaerobic/Environmental Chamber (Sheldon Manufacturing).

[0147]Genome sequencing and assembly. Whole-genome shotgun sequencing was carried out at the US Department of Energy Joint Genome Institute (JGI). Sequences were assembled into 227 contigs using the Phred/Phrep/Consed software and the sequence coverage was 9.2-fold. Gap closure and genome sequence finishing was carried out at Fidelity Systems using ThermoFidelase-Fimer direct genome sequencing technology (Slesarev et al., 2002, Proc Natl Acad Sci USA, 99:4644-4649). Shotgun reads with and without IS30 elements covering A5, A6 and A7 loci were identified and assembled separately. The presence and location of long repeated sequences in genomic DNA samples were verified by direct genomic sequencing of the unique/repeat junctions. The resolution of the most complex high GC-rich repeats was achieved by sequencing of PCR products amplified with a hybrid TopoTaq DNA polymerase with increased strand displacement capacity.

[0148]Bioinformatic analysis. Annotation of all open reading frames (ORFs) was carried out using Glimmer, GeneMark, JGI annotation tools and GAMOLA (Altermann et al., 2003, OMICS 2003, 7:161-169), before manual checking of all predicted genes. A comparative analysis of the two B. longum genomes was conducted using MUMmer3, ACT4 and ClustalX. The origin of replication and terminus were predicted using OriLoc (Frank and Lobry, 2000, Bioinformatics 2000, 16:560-561). Codon usage was analyzed using the General Codon Usage Analysis (GCUA) program (McInerney 1998, Bioinformatics 1998, 14:372-373). The base-deviation index (BDI) was performed by scaled χ2 analysis of Artemis8. To predict gene functions, the two conserved protein domain databases of GAMOLA and InterProScan were used. COG functional categories were used for functional classification of all genes in both B. longum genome sequences.

[0149]Molecular techniques. General sequencing was conducted using a Big-Dye terminator and ABI Prism 3730x1 Auto sequencer (Applied Biosystems). All PCR primers are listed in Table 5. For Southern blot analysis of unique region 12, a 646 bp probe from the lanM gene was obtained using PCR with LANT-F and LANT-R primers. Probes for IS elements were also PCR amplified. Probes were DIG-labeled and hybridized with digested genomic DNA according to the manufacturer's instructions (Roche). Pulsed field gel electrophoresis of XbaI-digested B. longum genomes was performed using a CHEF-DR III Variable Angle Pulsed Field Electrophoresis System according to manufacturer's instructions (Bio-Rad).

TABLE-US-00005 TABLE 5 Primers used. Target region Unique region no.^a Primer Sequence^b Size Reference Oligo cluster 15 OLIGO15-F 5'-GAAATCCCGAAANACNACC-3' 1,793 bp This study OLIGO15-R 5'-GTTGCCGATGTTYTGNCC-3' 6 OLIGO6-F 5'-GTATGTGATGAGCGGNAGY-3' 1,840 bp This study OLIGO6-R 5'-ACCAACGGATTTYTGNGG-3' 9 OLIGO9-F 5'-AAGTTCACCGATGARACN-3' 2,001 bp This study OLIGO9-R 5'-GTAACGCAACGARTAYTCC-3' 11 OLIGO11-F 5'-TCCCCAACTACATTATHGTNG-3' 1,419 bp This study OLIGO11-R 5'-TCAACACCATCNGCNACC-3' Arsenic cluster 5 ARS5-F 5'-ATTGGCTTATTGCTNACN-3' 736 bp This study ARS5-R 5'-GACTGCTTCAACTGNAGDATCC-3' 7 ARS7-F 5'-ACAGTCCCAATACAGTAARACN-3' 1,125 bp This study ARS7-R 5'-CTCAAAGAAATTAGANGCNCC-3' Lantibiotic 12 LANT-F 5'-CGCTATTACACCAGATACG-3' 646 bp This study LANT-R 5'-GGTAGACATACAGGTTCTCC-3' Positive control (16S rRNA gene) 16S-F 5'-CAGCWGCCGCGGTAATWC-3' 890 bp (Lane et al., 16S-R 5'-ACGGGCGGTGTGTRC-3' 1985) Deletion of lantibiotic operon Forward F3 5'-ATCCAACGAGCAAGAACC-3' This study Reverse R3 5'-GTGAAATCACCACTACCACC-3' Deletion of MIC III region Upstream MIC-F1 5'-CACATCTTGGAACTGCTTGG-3' This study MIC-R1 5'-CGTACACCGATGAATGACC-3' Downstream MIC-F2 5'-GTTCTTCGTCACCTCCACC-3' MIC-R2 5'-AGTAATGTCCCGAATCCTCC-3' IS elements IS30 IS30-F 5'-GACAAACCCAAGACCCTCC-3' 352 bp This study IS30-R 5'-CGTGCATATCCCCATTATCC-3' IS21 IS21-F 5'-GCCCCAAGTACAGTCTATCC-3' 681 bp This study IS21-R 5'-CAGAACGAACAATCGAACC-3' IS256 IS256-F 5'-TGTCACAGCAGATTCTACAGG-3' 719 bp This study IS256-R 5'-CAGCAATTCGTTCACAGC-3' ISL3 ISL3-F 5'-CGAGATCGTCGAGCTTTCC-3' 169 bp This study ISL3-R 5'-ATCAGGGCGATGAGGTTGG-3' ^aas defined in the text; ^bY (C/T), R (A/G), H (A/C/T), D (A/G/T), N (A/T/G/C), W (A/T) SEQ ID NOs: 26-55 Lane et al., 1985. Proc. Natl. Acad. Sci. USA 82: 6955-6959.

[0150]Identification of gene homologs between the two B. longum genomes. Comparative nucleotide substitution analysis by Nei and Gojobori's algorithm (Nei and Gojobori, 1986, Mol Biol Evol 1986, 3:418-426) was used to identify gene homologs. The predicted genes of both genome sequences were compared using the local BlastN program in the NCBI toolkit and 1,590 aligned genes were used for the nucleotide substitution analysis by Nei's unweighted method I (Nei and Gojobori, 1986, Mol Biol Evol 1986, 3:418-426). According to the ratio of dN:dS, all matched genes were categorized into three groups, highly conserved (<0.035), normal, and positive selection (>1).

[0151]Minimal inhibitory concentration of arsenic. To determine the minimal inhibitory concentration of arsenic, BLIM was supplemented with different concentrations of sodium arsenite (AsO2-, 1 to 100 mM) and sodium arsenate (AsO3-, 1 to 500 mM). Freshly grown cultures were sub-inoculated into the arsenite/arsenate media and incubated anaerobically at 37° C. for 48 hours.

[0152]Adaptation of B. longum DJO10A to in vitro fermentation conditions. B. longum DJO10A was grown in BFM continuously up to ˜1,000 generations. The culture was then serially diluted and plated on BFM agar. Ten colonies were randomly selected for analysis.

[0153]Mapping the deletions in strain DJO10A-JH1. To find the precise location of the deletion of the lantibiotic operon in the B. longum DJO10A-JH1 genome, PCR was used to test for several genes within the lantibiotic operon. The two primers F3 (position 1,974,570-1,974,587 bp) and R3 (position 1,996,024-1,996,005 bp) were used to amplify a ˜1.8 kb region spanning the deletion and sequencing located the precise borders (FIG. 6). To map the position of the deletion in the 140.7 kb XbaI fragment, primers MIC-F1 (position, 1,539,767-1,539,768) and MIC-R1 (position, 1,542,535-1,542,553) were used to amplify the upstream region of MIC III and primers MIC-F2 (position, 1,543,406-1,543,424) and MIC-R2 (position, 1,545,713-1,545,732) were used to amplify the downstream region.

[0154]Bioassay for lantibiotic activity. B. longum DJO10A was inoculated into the center of an MRS agar plate and incubated anaerobically at 37° C. for 2 days. After incubation, molten 0.5% top agar of the same medium containing 1% of an indicator strain was overlaid on the plates prior to incubation.

[0155]Simulated fecal competitive analysis of bifidobacteria. To access the competitive `fitness` of the wild-type B. longum DJO10A compared to its in vitro adapted derivative strain DJO10A-JH1, a simulated in vitro fecal system was developed. Triplicate experiments for each strain were used. Each experiment was conducted in 10 g sterilized feces in an anaerobic chamber, to which 0.38 g Reinforced Clostridial Medium (RCM) and 0.02 g mucin (Porcine gastric type III) was added. The two competitor bacteria were added to all tubes at calculated concentrations of 1.2×10⁷ cfu/g for E. coli DJOec1 and 5.1×10⁷ for Clostridium difficile DJOcd1. B. longum DJO10A was added to three tubes at a calculated concentration of 4.0×10⁷ cfu/g and strain DJO10A-JH1 to the other three tubes at 4.4 cfu/g. Standard viable plate counts were used to calculate all bacterial concentrations. After thorough mixing in an anaerobic environment, the tubes were left at 37° C. for 3 days, whereby the entire fecal samples were homogenized in 90 ml peptone water to conduct an accurate serial plate count analysis.

Example 2

Preparation of Extracted Bifidobacterium Lantibiotic

[0156]B. longum strain DJO10A was grown in MRS broth supplemented with 0.05% L-cysteine.HCl (Sigma) or Bifidobacteria Low-Iron Medium (BLIM). The broth was then used to cover the surface of an MRS agar plate supplemented with 100 mM PIPES or a BLIM agar plate supplemented with 100 mM PIPES. The plates were incubated under anaerobic conditions using either the BBL Anaerobic system (BBL) or the Bactron II Anaerobic/Environmental Chamber (Sheldon Manufacturing) at 37° C. for 2 days. Twenty plates were used.

[0157]The cells and agar medium were crushed and the mixture was extracted with 95% methanol using routine methods. The extraction was allowed to proceed overnight. The final volume was placed in a SpeedVac to remove the methanol and concentrate the lantibiotic.

[0158]The remaining agar was removed by size fractionation using Millipore CentriPrep filtration for partial purification. The extract was fractionated with a Centriprep-30 (30 kDa cut-off) by centrifugation twice at 1,500×g for 15 minutes and 10 minutes, respectively, and the filtrate (<30 kDa) transferred to a Centriprep-10 (10 kDa cut-off). This was subjected to centrifugation twice at 3,000×g for 40 minutes and 10 minutes, respectively. The filtrate was transferred to a Centriprep-3 (3 kDa cut-off). This was subjected to centrifugation twice at 3,000×g for 95 minutes and 35 minutes, respectively. The fractionated solution (3-10 kDa) was collected and concentrated by SpeedVac machine.

[0159]The concentrated lantibiotic was resuspended, and immediately tested using a diffusion method. Agar plates were made with MRS or BLIM and supplemented with PIPES, and a well of 5 millimeters cut into the middle of each plate. One hundred microliters of the suspended lantibiotic were placed in the well and allowed to diffuse until the liquid in the well was gone. The plates were then overlayed with the indicator strain.

[0160]The lantibiotic inhibited the growth of the indicator strains M. leuteus, L. lactis, S. aureus, S. epidermdis, E. coli, S. marcescens, and P. vulgaris. The lantibiotic did not inhibit P. aeuruginosa in this assay; however, it is not possible to conclude from these data that the lantibiotic will not inhibit P. aeuruginosa.

Example 3

Heat Resistance of Extracted Bifidobacterium Lantibiotic

[0161]The lantibiotic from example 2 was placed in a boiling water bath for 10 minutes and then tested for activity using the diffusion method and M. leuteus as the indicator strain. The lantibiotic was active after boiling for 10 minutes.

Example 4

Proteolytic Analysis of the Extracted Bifidobacterium Lantibiotic

[0162]Stock solutions of the proteolytic enzymes were prepared as follows.

[0163]Pepsin (Sigma No. P6887) was dissolved in 2 mM Tris.HCl or water at pH 2, 37° C., at a concentration of 34600 U/ml (10 mg/ml).

[0164]Pronase E (Sigma No. P5147) was dissolved in 20 mM Tris.HCl or 50 mM phosphate buffer at pH 7.5, 37° C., at a concentration of 5500 U/ml (500 mg/ml).

[0165]α-Chymotrypsin (Sigma No. C4129) was dissolved in 80 mM Tris.HCl at pH 7.8, 25° C., at a concentration of 5100 U/ml (100 mg/ml).

[0166]Proteinase K (Sigma P2308) was dissolved in 10 mM Tris.HCl at pH 7.5, 37° C., at a concentration of 6000 U/ml (200 mg/ml).

[0167]Trypsin (MP biochemical (ICN) No. 15021310), pH 7.6, 25° C., was used at the concentration 4750 U/ml (50 mg/ml).

[0168]Thermolysin (Fluka No. 88303), pH 7.2, 37° C., was used at the concentration 6000 U/ml (150 mg/ml).

[0169]One hundred microliters of the lantibiotic from example 2 was used in each assay. The proeolytic enzymes were added to separate 100 mls of the lantibiotic as follows: pepsin, 5 μl (173 U); pronase E, 20 μl (110 U); α-Chymotrypsin, 20 μl (102 U); proteinase K, 20 μl (120 U); trypsin, 20 μl (95 U); and thermolysin, 20 μl (120 U). Samples containing pepsin, Pronase E, Proteinase K, or Thermolysin were incubated at 37° C., and samples containing α-Chymotrypsin or Trypsin were incubated at 25° C. The incubation was for 24 hours. After digestion, the sample was neutralized to pH7.5, and all samples were incubated in boiling water for 10 minutes to remove the proteolytic enzyme activity.

[0170]Each sample was tested for activity using the diffusion method, 50 μl of the sample containing the inactivated proteolytic enzyme, and M. leuteus as the indicator strain. The extracted lantibiotic was sensitive to pepsin (at pH 2) and to pronase E (at pH7.5), and insensitive to the other 4 proteolytic enzymes.

[0171]The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

[0172]Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0173]Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0174]All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Sequence CWU 1

4411805DNAinfluenza A virus 1ttattcgtct tcagggagca aaagcagggg ttataccata gacaaccgaa caaagacaat 60gaccatcact tttctcatcc tcctgttcac agtagtgaaa ggggaccaaa tatgcatcgg 120ataccatgcc aacaattcca cagaaaaagt tgacacaatc ttggaacgaa acgtcaccgt 180gactcatgcc aagaacattc ttgaaaagac gcataatgga aagttgtgca gattgagcgg 240gatccctcca ttggaactgg gggattgcag cattgcaggt tggctccttg gaaatccgga 300atgtgaccgg ctcttaagtg tacctgaatg gtcctatata gtggaaaagg aaaacccggt 360gaatggtctg tgctatccag gcagtttcaa tgattatgag gaattgaaac atcttctcac 420cagtgtgaca cactttgaga aagttaagat tctgcccaga gatcaatgga cccagcacac 480aacaactggt ggttctcggg cctgtgcagt atctggaaac ccgtcattct ttaggaacat 540ggtttggctt acaaagaaag ggtcaaacta cccaattgct aaaaggtcat acaacaacac 600aagtggggag caaatgctgg taatatgggg gatacatcac cccaatgacg atgcggaaca 660gaggacactg taccagaatg tgggaacata tgtttccgtt ggaacatcaa cactaaataa 720gaggtcaatc cctgaaatag caacaaggcc caaagtcaat ggactgggag gaagaatgga 780attctcttgg actctattgg agacatggga tgtcataaat tttgagagca ctggtaattt 840aattgcacca gaatacggat tcaaaatatc aaagagagga agctcaggaa ttatgaagac 900agagaaaata cttgaaaatt gtgaaaccaa atgtcagacc cccttggggg caataaatac 960aacattgccc tttcacaaca ttcacccatt gacaataggt gagtgcccca agtatgtaaa 1020gtcagataga ctgattttgg cgacaggact aagaaatgtc ccccagattg aatcaagggg 1080attgtttgga gcaatagctg ggtttataga aggcggatgg caagggatgg ttgatggctg 1140gtatgggtac catcacagca atgatcaagg atcaggatat gcagcagaca aagaatccac 1200tcaaaaggca attgatggga taactaacaa agtaaattct gtgattgaaa agatgaacac 1260tcagtttgag gctgttggga aagagttcaa caacctagag agaagactgg aaaacttaaa 1320taaaaagatg gaagatggat ttattgatgt atggacatat aatgccgaac tcctagttct 1380aatggaaaat gagaggacac ttgatttcca tgattctaat gtgaagaatc tgtacgataa 1440ggtcagaatg caattgagag acaatgctaa ggaaataggg aacggatgct ttgagtttta 1500tcataaatgt gatgatgaat gcatgaatag tgtcaggaat gggacatatg attatatcaa 1560atatgaggaa gagtccaagc tgaacaggaa cgaaatcaaa ggagtgaaat tgagcaatat 1620gggggtttat caaatacttg ctatatacgc tacagttgca ggctctttgt cactggcaat 1680catgatagct gggatttctt tctggatgtg ttctaatggg tctctgcaat gcagaatttg 1740catatgactg taagtcaatt tgtaattaaa aacacccttg tttctactaa tacgagacga 1800tataa 18052989DNAinfluenza A virus 2ttaaagatga gtcttctaac cgaggtcgaa acgtatgttc tctctatcgt cccgtcaggc 60cccctcaaag ccgagatagc acagagactc gaagacgttt ttgcagggaa aaacaccgat 120cttgaggctc tcatggaatg gctaaagaca agaccaatcc tgtcacctct gactaagggg 180attttagggt ttgtgttcac gctcaccgtg cccagtgagc gaggactgca gcgtagacgt 240tttgttcaga atgccctcaa tgggaatggt gacccgaaca acatggacaa ggcggtcaaa 300ctttacagga aactaaaaag ggaaataaca ttccatgggg ccaaagaagt agcgctcagt 360tactctgctg gtgcacttgc cagttgcatg ggcctcatat acaacagaat gggaactgtc 420accactgagg ttgcctttgg tctggtatgc gcaacctgtg aacagattgc tgattctcag 480catcgatccc atagacaaat ggtgacaaca accaatccac taatcaggca cgagaacaga 540atggtgatag ccagcacaac agctaaagca atggaacaaa tggctggatc aagcgaacaa 600gcagcagagg ctatggaggt tgccagccag gctagacaaa tggtacaggc aatgagaaca 660attgggactc accctagttc cagcactggt ctaaaagatg atcttcttga aaatttacag 720gcctatcaga agcggatggg agtgcaaatg caacgattca aatgatcctc tcattgatgc 780tgcaagcatc attgggattt tgcacctgat attgtggatt cttgatcgtc tttttttcaa 840atgcatttac cgtcgcttta aatacggtct gcaaagaggg ccttctacgg aaggagtgcc 900ggagtccatg agggaagaat atcgacagaa acagcagagt gctgtggatg ttgacgatgg 960tcattttgtc aacatagtgc tagagtaaa 98931440DNAinfluenza A virus 3cgagatgaat ccgaatcaga agataataac aatcggggta gtgaatacca ctctgtcaac 60aatagccctt ctcattggag tgggaaactt agttttcaac acagtcatac atgagaaaat 120aggagaccat caaatagtga cctatccaat aataacgacc cctgcagtac cgaactgcag 180tgacactata ataacataca ataacactgt gataaacaac ataacaacaa caataataac 240tgaagaagaa aggcctttca agtctccact accgctgtgc cccttcagag gattcttccc 300ttttcacaag gacaatgcaa tacgactggg tgaaaacaaa gacgtcatag tcacaagaga 360gccttatgtt agctgcgata atgacaactg ctggtccttt gctctcgcac aaggagcatt 420gctagggacc aaacatagca atgggaccat taaagacagg acaccatata ggtctctaat 480tcgtttccca ataggaacag ctccagtact aggaaattat aaagagatat gcattgcttg 540gtcgagcagc agttgctttg acgggaaaga gtggatgcat gtgtgcatga cagggaacga 600taatgatgca agtgcccaga taatatatgg agggagaatg acagactcca ttaaatcatg 660gagaaaggac atactaagaa ctcaggagtc tgaatgccaa tgcattgacg ggacttgtgt 720tgttgctgtc acagatggcc ctgctgctaa tagtgcagat tacagggttt actggatacg 780ggagggaaaa ataataaagt atgaaaatgt tcccaaaaca aagatacaac acttagaaga 840atgttcctgc tatgtggaca ttgatgttta ctgtatatgt agggacaatt ggaagggctc 900taacagacct tggatgagaa tcaacaacga gactatactg gaaacagggt atgtatgtag 960taaattccac tcagacaccc ccaggccagc tgacccttca acaatgtcat gtgactcccc 1020aagcaatgtc aatggaggac ccggagtgaa ggggtttggt ttcaaagctg gcgatgatgt 1080atggttaggt agaacagtgt cgactagtgg tagatcgggc tttgaaatta tcaaagttac 1140agaagggtgg atcaactctc ctaaccatgt caaatcaatt acacaaacac tagtgtccaa 1200caatgactgg tcaggctatt ccggtagctt cattgtcaaa gccaaggact gttttcagcc 1260ctgtttttat gttgagctta tacgagggag gcccaacaag aatgatgacg tctcttggac 1320aagtaatagt atagttactt tctgtggact agacaatgaa cctggatcgg gaaattggcc 1380agatggttct aacattgggt ttatgcccaa gtaatagaaa aaagcacctt gtttctacta 144041532DNAinfluenza A virus 4acccatcaat gagtgacatc gaagccatgg cgtctcaagg caccaaacga tcatatgaac 60aaatggagac tggtggggaa cgccaggatg ccacagaaat cagagcatct gtcggaagaa 120tgattggtgg aatcgggaaa ttctacatcc aaatgtgcac tgaactcaaa ctcagtgact 180atgagggacg actaatccaa aatagcatga caatagagag aatggtgctc tctgcttttg 240atgagagaag aaataaatac ctagaagagc atcccagtgc agggaaggat cctaagaaaa 300ctggaggacc catatataga agagtagacg gaaagtggat gagagaactc attctttatg 360acaaagaaga aataaggaga gtttggcgcc aagcaaacaa tggtgaagat gcaacagctg 420gtcttgctca tatcatgatt tggcactcca atctgaatga tgccacgtac cagagaacaa 480gagcgcttgt tcgcaccgga atggatccca gaatgtgctc tctaatgcaa ggttcaacac 540ttcccagaag gtctggggcc gcaggtgctg cagtgaaagg agttggaaca atagcaatgg 600aattaatcag aatgatcaaa cgtgggatca atgaccgaaa cttctggaga ggtgaaaatg 660gacgaaagac aaggattgca tatgaaagaa tgtgcaatat tctcaaggga aaatttcaga 720cagctgccca gagggcaatg atggatcaag tgagagaaag tcggaacccc ggaaacgctg 780agattgaaga tctcattttc ctggcacggt cagcacttat tctaagggga tcagttgcac 840ataagtcttg cctgcctgct tgtgtgtatg ggcttgcagt ggcaagtggg catgactttg 900aaagggaagg gtattcactg gtcgggatag acccatttaa attactccaa aacagtcaag 960tgttcagctt gataagacca aatgaaaacc cagctcacaa gagtcaatta gtgtggatgg 1020catgccactc tgctgcattt gaggatctga gggtatcaag tttcatcaga gggaagaaag 1080tgattccaag aggaaggctc tccacaagag gggttcagat tgcttcaaat gagaatgtgg 1140aagccatgga ttccaatacc ttagagctga gaagcagata ctgggccata aggaccagaa 1200gtggaggaaa taccaatcaa cagaaggcat ccgcgggcca gatcagtgtg caacccacat 1260tctcagtgca acggaatctc ccttttgaaa gagcaaccgt tatggcagct ttcagcggga 1320acaatgaagg acggacatcc gatatgcgaa cagaagttat aaggatgatg gaaaatgcaa 1380agccagaaga tttgtccttc caggggcggg gagtcttcga gctctcggac gaaaaagcaa 1440cgagcccgat cgtgccttcc tttgacatga gtaatgaagg atcttatttc ttcggagaca 1500atgcagagga gtatgacagt tgaggaaaaa ta 15325851DNAinfluenza A virus 5caaaaagaca taatggactc cacaactgtg tcaagctttc aggtagactg tttcctttgg 60cacatccgca aacggtttgc agacaatgga ttgggtgatg ccccattcct tgatcggctc 120cgccgagatc aaaagtctct aaaaggaaga ggcaacaccc ttggcctcga cattgaaaca 180gccactcttg ttgggaaaca aattgtggag tggattttga aagaagaatc cagcgataca 240cttaagatga ctattgcatc tgtacctact tcgcgctatt tagctgacat gaccctcgag 300gaaatgtcac gagactggtt aatgctcatg cctaggcaaa agataatagg ccctctttgt 360gtgcgaatgg accaggcgat catggaaaag aacatcatac tgaaagcgaa cttcagtgtg 420atctttaacc gattagagac tttgatacta ctaagggctt tcacagagga gggaacaatc 480gttggagaaa tttcacaatt accttctctt ccaggacata ctaatgagga tgtcaaaaat 540gcaattgggg tcctcatcgg aggacttgaa tggaatggta acacggttcg aggctctgaa 600aatctacaga gattcgcttg gagaaaccgt aatgaggatg ggagaccttc actacctcca 660gagaagaaat gaaaagtggc gagagcaatt gggacaaaaa tttgaggaaa taaggtggtt 720aattgaagaa gtgcggcaca gattgaaaac gacagagaat agttttgaac aaataacatt 780catgcaagcc ttacaactac tgcttgaagt agaacaagag ataaggactt tctcgtttca 840gcttatttaa t 85162230DNAinfluenza A virus 6ccaaaatgga agactttgtg cgacaatgct tcaatccaat gatcgtcgag cttgcggaaa 60aggcaatgaa ggaatatggg gaagatccaa aaatcgaaac taacaaattc gcagcaatat 120gcactcactt ggaagtatgt ttcatgtatt cggatttcca cttcattgat gagcggggcg 180aatcaataat tgtggaatct ggtgatccaa atgcattact gaagcaccga tttgaaataa 240ttgaaggaag agaccgaaca atggcctgga cagtggtgaa tagcatctgc aacaccacag 300gggtcgagaa gcctaaattt cttccggatc tgtatgatta caaggagaac cgattcattg 360aaattggagt aacacggaga gaggttcata tatactacct agagaaagcc aacaagataa 420aatctgagaa gacacacatt cacatctttt catttactgg agaagaaatg gccaccaaag 480cagactacac tcttgatgaa gaaagcaggg caagaatcaa aaccaggcta ttcactataa 540gacaagaaat ggccagcagg ggtctatggg attcctttcg tcagtctgaa agaggcgaag 600agacaattga ggaaagattt gaaatcacag gaaccatgcg taggcttgcc gaccaaagtc 660tcccaccgaa cttctccagc cttgaaaact ttagagccta tgtggatgga ttcgaaccga 720acggctgcat tgagggcaag ctttctcaaa tgtcaaaaga agtgaatgcc aggattgagc 780cattcctgaa gacaacacca cgccctctca aattacctga tgggccccct tgctctcagc 840ggtcaaaatt cttgctgatg gatgccttga aactaagcat cgaagacccg agtcacgagg 900gagagggtat accactatac gatgcaatca aatgcatgaa gacatttttc ggctggaaag 960agcccaacat aatcaaacca catgagaaag gcataaatcc caattacttt ctggcttgga 1020agcaagtgct agcagagctc caggaccttg aaaatgaaga gaagatccca aagacaaaga 1080acatgaagaa aacaagccaa ttgaagtggg cgcttggtga gaatatggca ccagaaaaag 1140tggactttga ggattgcaag gacattggcg atctaaagca gtatgatagt gatgagccag 1200agcctagatc gctggcaagc tggatccaga gtgaattcaa taaggcatgt gaattgaccg 1260actcgagctg gatagaactt gatgaaatag gagaagatgt tgctccgatt gaacacattg 1320caagtatgag gaggaactat tttacagcag aagtgtccca ctgcagggcc actgaataca 1380taatgaaagg agtctacata aatacagctc tgctcaatgc atcttgtgca gccatggatg 1440acttccagct gattccaatg ataagcaaat gtagaacaaa ggaaggaaga cggaaaacaa 1500acctgtatgg gttcatcata aaaggaagat ctcatttgag gaatgatact gatgtggtaa 1560actttgtgag catggaattt tctctcactg acccgaggct agaaccacac aaatgggaga 1620agtattgtgt tcttgaaata ggagatatgc tcctgaggac tgcaataggc caagtgtcaa 1680ggcccatgtt cctgtacatt agaaccaatg ggacctccaa gatcaagatg aaatggggta 1740tggaaatgag gcgctgcctc cttcaatctc ttcaacagat tgagagcatg attgaggctg 1800agtcttctgt caaagaaaag gacatgacta aggaattctt tgaaaacaag tcggaaacgt 1860ggccaattgg agaatccccc agaggagtag aggaaggctc tattgggaaa gtatgcagaa 1920ccttactggc aaagtctgta ttcaacagtc tgtacgcatc tccacaactt gaggggtttt 1980cagctgaatc gagaaaattg cttctcattg ttcaggcact tagggacaac ctggaacctg 2040ggaccttcga tcttgggggg ctatacgaag caattgagga gtgcctgatt aatgatccct 2100gggttttgct taatgcatct tggttcaact ccttcctcac acatgcactg aaatagttgt 2160ggcaatgcta ctatttgcta tccatactgt ccaaaaaagt accttgtttc tactaataga 2220agagcgatga 223072339DNAinfluenza A virus 7ttgaatggat gtcaatccga ccctactttt cctaaaagtt ccagcgcaaa atgccataag 60caccacattc ccttatactg gagatcctcc atacagccat ggaacaggaa caggatacac 120catggacaca gtcaacagaa cacatcaata ttcagaaaaa gggaagtgga cgacaaacac 180tgagactggg gcaccccaac tcaatccaat tgatggacca ctacctgagg ataatgaacc 240aagtgggtat gcacagacag actgtgttct agaggccatg gctttccttg aagaatccca 300cccagggata tttgagaatt catgccttga aacaatggaa gttgttcaac aaacaagggt 360ggataaacta actcaaggtc gccagactta tgattggaca ttaaacagaa atcaaccagc 420agcaactgca ttggccaaca ccatagaagt ttttaggtcg aatagtctaa cagccaatga 480gtcaggaaga ctaataaatt tcctaaagga tgtaatggaa tcaatggata gagaagaagt 540ggagataaca acacactttc gaagaaaaag gagagtgaga gacaacatga ccaagaagat 600ggtcacacaa agaacaatag gaaagaaaaa acaaagattg agtaagagaa gttatctaat 660aagagcactt acattgaata cgatgaccaa agatgcagag agaggcaaat taaaaagaag 720ggctatcgca acacccggga tgcaaattag agggttcgtg tactttgttg agactttagc 780taggagcatt tgcgaaaagc ttgaacagtc tggactccca gtagggggaa atgaaaagaa 840ggccaaattg gcaaatgttg tgagaaaaat gatgactaat tcacaagaca cagagctttc 900tttcacaatc actggggaca atactaagtg gaatgaaaat caaaatcctc gaatgttcct 960ggcgatgatt acatatatca cccgaaatca acccgagtgg ttcagaaaca tcctgagcat 1020ggcacccata atgttttcaa acaaaatggc aagactagga aaagggtaca tgttcgagag 1080taaaaggatg aagctccgaa cacaaatacc agcagaaatg ctagcaagca ttgatctgaa 1140gtatttcaat gaatcaacaa ggaagaaaat tgagaaaata aggcctcttc taatagatgg 1200cacagcatca ttgagccctg gaatgatgat gggcatgttc aacatgctaa gtacggtttt 1260gggagtctcg atactgaatc ttggacaaaa gaaatacacc aggacaacat actggtggga 1320tgggctccaa tcctccgacg attttgccct cattgtgaat gcaccaaatt atgagggaat 1380acaagcagga gtgaatagat tctacaggac ctgcaagtta gtaggaataa acatgagcaa 1440aaagaagtcc tatataaata aaacagggac atttgaattc acaagctttt tttatcgcta 1500tgggtttgtg gctaatttta gcatggagct gcccagtttt ggagtgtctg gaataaatga 1560atcagctgat atgagtattg gagtaacagt gataaagaac aatatgataa acaatgatct 1620tggacctgca acagcccaga tggcccttca attgttcatc aaagactaca gatacacata 1680taggtgccac agaggagaca cacaaattca gacgagaaga tcattcgagc taaagaagct 1740atgggatcaa acccgatcaa atgcaggact attagtatct gatggaggac caaacttata 1800caatatcagg aatcttcaca ttcctgaagt ctgcttaaaa tgggagctaa tggatgagga 1860ttatcgggga aggctttgta atcccctgaa tccttttgtc agccataaag agattgattc 1920tgtaaacaat gctgtggtga tgccagccca tggcccagcc aaaagcatgg aatatgatgc 1980cgttgcaact acacactcct ggatccccaa gaggaatcgc tctattctca acacaagcca 2040aaggggaatt cttgaggatg aacagatgta ccagaagtgc tgcaacctgt tcgagaaatt 2100tttccctagt agttcataca ggagaccggt tggaatttct agcatggtgg aggccatggt 2160gtctagggcc cggattgatg ccagaatcga cttcgagtct ggacggatta agaaagaaga 2220gttctctgag atcataagga tctgttccac cattgaagaa ctcagacggc aaacatgatg 2280aatttggctt gtccttcatg aaaaaatgcc ttgtttctac taatacgaga cgatataaa 233982299DNAinfluenza A virus 8tatattcaat atggagagaa taaaagaact aagagaccta atgtcgcagt cccgcactcg 60cgaaatactc accaagacca ctgtggacca tatggccata atcaaaaagt acacatcggg 120aaggcaagag aagaaccctg cactcagaat gaagtggatg atggcaatga agtacccaat 180cacagcagac aagagaataa tggacatgat tccagagaga aatgaacaag gacaaaccct 240ctggagcaaa acaaacgatg ctggatcgga ccgcgtgatg gtatcacccc tggctgtaac 300atggtggaat aggaatggcc caacagcaag cacagttcac taccctaagg tatataaaac 360ttatttcgaa aaagtcgaaa ggttaaaaca tggtaccttt ggccctgtcc acttcaggaa 420tcaagttaaa ataagaagga gagttgacac aaaccctggt cacgcagatc tcagagccaa 480ggaggcacag gatgtgatca tggaagttgt tttcccaaat gaagtggggg caagaatact 540gacatcagag tcacagctga caataacaaa agagaagaaa gaagagctcc aggattgtaa 600aattgctccc ttaatggtgg catacatgct agaaaaagag ttggtccgta aaacgaggtt 660tctcccggtg gctggtggaa caggcagtgt ttatattgaa gtgctgcatt taactcaggg 720gacatgctgg gagcaaatgt acactccagg aggagaagtg agaaatgatg atgttgacca 780aagtttgatt attgctgcta gaaacatagt aagaagagca gcagtgtcag cagacccatt 840agcatctctt ttggaaatgt gccacagcac acagattgga ggaataagga tggtggacat 900ccttagacag aatccaacgg aggaacaagc cgtagacata tgcaaggcag caatggggct 960gaggattagc tcctctttca gctttggtgg gttcactttc aaaagaacaa gtgggtcatc 1020agttaagaga gaagaagaag tgctcacggg caaccttcaa acactgaaaa taagagtaca 1080tgaaggatat gaagaattca caatggtcgg gagaagagca acagctattc tcagaaaagc 1140aaccaggaga ttgatccagt tgatagtaag tgggagagac gagcagtcaa ttgctgaggc 1200aataattgtg gccatggtat tttcacaaga ggactgcatg atcaaggcag ttaggggcga 1260tctgaacttt gtcaataggg caaaccagcg actgaatccc atgcatcaac tcttgaggca 1320tttccaaaaa gatgcaaaag tgcttttcca gaattgggga attgaaccca tcgacaatgt 1380gatgggaatg atcgggatat tgcccgatat gaccccaagc acggagatgt cgctgagagg 1440gataagagtc agcaaaatgg gagtagatga atactccagc acggagagag tggtagtgag 1500cattgacaga tttttgaggg ttcgggatca acgagggaac gtactattgt ctcctgaaga 1560ggtcagtgag acacaaggga cggagaaatt ggcaataact tattcgtcat cgatgatgtg 1620ggagatcagt ggccctgagt cagtgctggt caacacttat caatggatca taaggaattg 1680ggaaagtttg aaaattcaat ggtcacaaga tcccacgatg ttatacaaca aaatggaatt 1740tgaaccattt cagtctcttg tccctaaagc aaccagaagt cgttacagtg ggttcgtgag 1800gacactgttc cagcaaatgc gggatgtgat tggaacattt gacactgtcc aaataataaa 1860acttctcccc tttgctgctg ctccaccaga acagagtagg atgcagtttt cctcactgac 1920tgtgaatgtg agaggatcag ggttgaggat actggtaaga ggcaattctc cagtattcaa 1980ttacaataaa gcaaccaaaa ggcttacagt tcttggaaag gatgcaggtg aattgaccga 2040agacccagat gaaggcacag ctggagtgga gtctgctgtc ctgaggggat ttctcatttt 2100gggcaaagaa gacaagagat atggtccagc attaagcatc aatgaactga gcaatcttgc 2160aaaaggagag aaagctaatg tgctaattgg gcaaggagac gtagtgttgg taatgaaacg 2220gaaacgggac tctagcatac ttactgacag ccagacagcg accaaaagaa ttcggatggc 2280catcaattag tgtcgaatt 22999562PRTinfluenza A virus 9Met Thr Ile Thr Phe Leu Ile Leu Leu Phe Thr Val Val Lys Gly Asp1 5 10 15Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val Asp20 25 30Thr Ile Leu Glu Arg Asn Val Thr Val Thr His Ala Lys Asn Ile Leu35 40 45Glu Lys Thr His Asn Gly Lys Leu Cys Arg Leu Ser Gly Ile Pro Pro50 55 60Leu Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro65 70 75 80Glu Cys Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile Val Glu85 90 95Lys Glu Asn Pro Val Asn Gly Leu Cys Tyr Pro Gly Ser Phe Asn Asp100 105 110Tyr Glu Glu Leu Lys His Leu Leu Thr Ser Val Thr His Phe Glu Lys115 120 125Val Lys Ile Leu Pro Arg Asp Gln Trp Thr Gln His Thr Thr Thr Gly130 135 140Gly Ser Arg Ala Cys Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn145 150 155 160Met Val Trp Leu Thr Lys Lys Gly Ser Asn Tyr Pro Ile Ala Lys Arg165 170 175Ser Tyr Asn Asn Thr Ser Gly Glu Gln Met Leu Val Ile Trp Gly Ile180 185

190His His Pro Asn Asp Asp Ala Glu Gln Arg Thr Leu Tyr Gln Asn Val195 200 205Gly Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys Arg Ser Ile210 215 220Pro Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Leu Gly Gly Arg Met225 230 235 240Glu Phe Ser Trp Thr Leu Leu Glu Thr Trp Asp Val Ile Asn Phe Glu245 250 255Ser Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys260 265 270Arg Gly Ser Ser Gly Ile Met Lys Thr Glu Lys Ile Leu Glu Asn Cys275 280 285Glu Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro290 295 300Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val305 310 315 320Lys Ser Asp Arg Leu Ile Leu Ala Thr Gly Leu Arg Asn Val Pro Gln325 330 335Ile Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly340 345 350Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn355 360 365Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala370 375 380Ile Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn385 390 395 400Thr Gln Phe Glu Ala Val Gly Lys Glu Phe Asn Asn Leu Glu Arg Arg405 410 415Leu Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Ile Asp Val Trp420 425 430Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu435 440 445Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Met450 455 460Gln Leu Arg Asp Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe465 470 475 480Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser Val Arg Asn Gly Thr485 490 495Tyr Asp Tyr Ile Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu500 505 510Ile Lys Gly Val Lys Leu Ser Asn Met Gly Val Tyr Gln Ile Leu Ala515 520 525Ile Tyr Ala Thr Val Ala Gly Ser Leu Ser Leu Ala Ile Met Ile Ala530 535 540Gly Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile545 550 555 560Cys Ile10252PRTinfluenza A virus 10Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro1 5 10 15Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe20 25 30Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr35 40 45Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe50 55 60Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val65 70 75 80Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys Ala85 90 95Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala100 105 110Lys Glu Val Ala Leu Ser Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met115 120 125Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe130 135 140Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg145 150 155 160Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu165 170 175Asn Arg Met Val Ile Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met180 185 190Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln195 200 205Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser210 215 220Ser Ser Thr Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr225 230 235 240Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys245 25011469PRTinfluenza A virus 11Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Val Val Asn Thr Thr1 5 10 15Leu Ser Thr Ile Ala Leu Leu Ile Gly Val Gly Asn Leu Val Phe Asn20 25 30Thr Val Ile His Glu Lys Ile Gly Asp His Gln Ile Val Thr Tyr Pro35 40 45Ile Ile Thr Thr Pro Ala Val Pro Asn Cys Ser Asp Thr Ile Ile Thr50 55 60Tyr Asn Asn Thr Val Ile Asn Asn Ile Thr Thr Thr Ile Ile Thr Glu65 70 75 80Glu Glu Arg Pro Phe Lys Ser Pro Leu Pro Leu Cys Pro Phe Arg Gly85 90 95Phe Phe Pro Phe His Lys Asp Asn Ala Ile Arg Leu Gly Glu Asn Lys100 105 110Asp Val Ile Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Asn Asp Asn115 120 125Cys Trp Ser Phe Ala Leu Ala Gln Gly Ala Leu Leu Gly Thr Lys His130 135 140Ser Asn Gly Thr Ile Lys Asp Arg Thr Pro Tyr Arg Ser Leu Ile Arg145 150 155 160Phe Pro Ile Gly Thr Ala Pro Val Leu Gly Asn Tyr Lys Glu Ile Cys165 170 175Ile Ala Trp Ser Ser Ser Ser Cys Phe Asp Gly Lys Glu Trp Met His180 185 190Val Cys Met Thr Gly Asn Asp Asn Asp Ala Ser Ala Gln Ile Ile Tyr195 200 205Gly Gly Arg Met Thr Asp Ser Ile Lys Ser Trp Arg Lys Asp Ile Leu210 215 220Arg Thr Gln Glu Ser Glu Cys Gln Cys Ile Asp Gly Thr Cys Val Val225 230 235 240Ala Val Thr Asp Gly Pro Ala Ala Asn Ser Ala Asp Tyr Arg Val Tyr245 250 255Trp Ile Arg Glu Gly Lys Ile Ile Lys Tyr Glu Asn Val Pro Lys Thr260 265 270Lys Ile Gln His Leu Glu Glu Cys Ser Cys Tyr Val Asp Ile Asp Val275 280 285Tyr Cys Ile Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Trp Met290 295 300Arg Ile Asn Asn Glu Thr Ile Leu Glu Thr Gly Tyr Val Cys Ser Lys305 310 315 320Phe His Ser Asp Thr Pro Arg Pro Ala Asp Pro Ser Thr Met Ser Cys325 330 335Asp Ser Pro Ser Asn Val Asn Gly Gly Pro Gly Val Lys Gly Phe Gly340 345 350Phe Lys Ala Gly Asp Asp Val Trp Leu Gly Arg Thr Val Ser Thr Ser355 360 365Gly Arg Ser Gly Phe Glu Ile Ile Lys Val Thr Glu Gly Trp Ile Asn370 375 380Ser Pro Asn His Val Lys Ser Ile Thr Gln Thr Leu Val Ser Asn Asn385 390 395 400Asp Trp Ser Gly Tyr Ser Gly Ser Phe Ile Val Lys Ala Lys Asp Cys405 410 415Phe Gln Pro Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Asn Lys420 425 430Asn Asp Asp Val Ser Trp Thr Ser Asn Ser Ile Val Thr Phe Cys Gly435 440 445Leu Asp Asn Glu Pro Gly Ser Gly Asn Trp Pro Asp Gly Ser Asn Ile450 455 460Gly Phe Met Pro Lys46512498PRTinfluenza A virus 12Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Gly1 5 10 15Gly Glu Arg Gln Asp Ala Thr Glu Ile Arg Ala Ser Val Gly Arg Met20 25 30Ile Gly Gly Ile Gly Lys Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys35 40 45Leu Ser Asp Tyr Glu Gly Arg Leu Ile Gln Asn Ser Met Thr Ile Glu50 55 60Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu65 70 75 80Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile85 90 95Tyr Arg Arg Val Asp Gly Lys Trp Met Arg Glu Leu Ile Leu Tyr Asp100 105 110Lys Glu Glu Ile Arg Arg Val Trp Arg Gln Ala Asn Asn Gly Glu Asp115 120 125Ala Thr Ala Gly Leu Ala His Ile Met Ile Trp His Ser Asn Leu Asn130 135 140Asp Ala Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp145 150 155 160Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser165 170 175Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Ile Ala Met Glu180 185 190Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg195 200 205Gly Glu Asn Gly Arg Lys Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn210 215 220Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp225 230 235 240Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu245 250 255Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His260 265 270Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala Val Ala Ser Gly275 280 285His Asp Phe Glu Arg Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe290 295 300Lys Leu Leu Gln Asn Ser Gln Val Phe Ser Leu Ile Arg Pro Asn Glu305 310 315 320Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala325 330 335Ala Phe Glu Asp Leu Arg Val Ser Ser Phe Ile Arg Gly Lys Lys Val340 345 350Ile Pro Arg Gly Arg Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn355 360 365Glu Asn Val Glu Ala Met Asp Ser Asn Thr Leu Glu Leu Arg Ser Arg370 375 380Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Lys385 390 395 400Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg405 410 415Asn Leu Pro Phe Glu Arg Ala Thr Val Met Ala Ala Phe Ser Gly Asn420 425 430Asn Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Val Ile Arg Met Met435 440 445Glu Asn Ala Lys Pro Glu Asp Leu Ser Phe Gln Gly Arg Gly Val Phe450 455 460Glu Leu Ser Asp Glu Lys Ala Thr Ser Pro Ile Val Pro Ser Phe Asp465 470 475 480Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr485 490 495Asp Ser13219PRTinfluenza A virus 13Met Asp Ser Thr Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp1 5 10 15His Ile Arg Lys Arg Phe Ala Asp Asn Gly Leu Gly Asp Ala Pro Phe20 25 30Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Lys Gly Arg Gly Asn35 40 45Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Leu Val Gly Lys Gln Ile50 55 60Val Glu Trp Ile Leu Lys Glu Glu Ser Ser Asp Thr Leu Lys Met Thr65 70 75 80Ile Ala Ser Val Pro Thr Ser Arg Tyr Leu Ala Asp Met Thr Leu Glu85 90 95Glu Met Ser Arg Asp Trp Leu Met Leu Met Pro Arg Gln Lys Ile Ile100 105 110Gly Pro Leu Cys Val Arg Met Asp Gln Ala Ile Met Glu Lys Asn Ile115 120 125Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Asn Arg Leu Glu Thr Leu130 135 140Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Thr Ile Val Gly Glu Ile145 150 155 160Ser Gln Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn165 170 175Ala Ile Gly Val Leu Ile Gly Gly Leu Glu Trp Asn Gly Asn Thr Val180 185 190Arg Gly Ser Glu Asn Leu Gln Arg Phe Ala Trp Arg Asn Arg Asn Glu195 200 205Asp Gly Arg Pro Ser Leu Pro Pro Glu Lys Lys210 21514716PRTinfluenza A virus 14Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu1 5 10 15Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asp Pro Lys Ile Glu Thr20 25 30Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr35 40 45Ser Asp Phe His Phe Ile Asp Glu Arg Gly Glu Ser Ile Ile Val Glu50 55 60Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu65 70 75 80Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn85 90 95Thr Thr Gly Val Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr100 105 110Lys Glu Asn Arg Phe Ile Glu Ile Gly Val Thr Arg Arg Glu Val His115 120 125Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His130 135 140Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp145 150 155 160Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe165 170 175Thr Ile Arg Gln Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg180 185 190Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr195 200 205Gly Thr Met Arg Arg Leu Ala Asp Gln Ser Leu Pro Pro Asn Phe Ser210 215 220Ser Leu Glu Asn Phe Arg Ala Tyr Val Asp Gly Phe Glu Pro Asn Gly225 230 235 240Cys Ile Glu Gly Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Arg245 250 255Ile Glu Pro Phe Leu Lys Thr Thr Pro Arg Pro Leu Lys Leu Pro Asp260 265 270Gly Pro Pro Cys Ser Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu275 280 285Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu290 295 300Tyr Asp Ala Ile Lys Cys Met Lys Thr Phe Phe Gly Trp Lys Glu Pro305 310 315 320Asn Ile Ile Lys Pro His Glu Lys Gly Ile Asn Pro Asn Tyr Phe Leu325 330 335Ala Trp Lys Gln Val Leu Ala Glu Leu Gln Asp Leu Glu Asn Glu Glu340 345 350Lys Ile Pro Lys Thr Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp355 360 365Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys370 375 380Lys Asp Ile Gly Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Pro385 390 395 400Arg Ser Leu Ala Ser Trp Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu405 410 415Leu Thr Asp Ser Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val420 425 430Ala Pro Ile Glu His Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala435 440 445Glu Val Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr450 455 460Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Asp Phe465 470 475 480Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg485 490 495Lys Thr Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg500 505 510Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr515 520 525Asp Pro Arg Leu Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu530 535 540Ile Gly Asp Met Leu Leu Arg Thr Ala Ile Gly Gln Val Ser Arg Pro545 550 555 560Met Phe Leu Tyr Ile Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys565 570 575Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile580 585 590Glu Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr595 600 605Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser610 615 620Pro Arg Gly Val Glu Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu625 630 635 640Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu645 650 655Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu660 665 670Arg Asp Asn Leu Glu Pro Gly Thr Phe Asp Leu Gly Gly Leu Tyr Glu675 680 685Ala Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala690 695 700Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Lys705 710 71515757PRTinfluenza A virus 15Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn1 5 10 15Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His20 25 30Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln35 40 45Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Thr Gly Ala Pro50 55 60Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser65 70 75 80Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu85 90 95Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu100 105 110Val Val Gln Gln Thr Arg Val Asp Lys Leu

Thr Gln Gly Arg Gln Thr115 120 125Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala130 135 140Asn Thr Ile Glu Val Phe Arg Ser Asn Ser Leu Thr Ala Asn Glu Ser145 150 155 160Gly Arg Leu Ile Asn Phe Leu Lys Asp Val Met Glu Ser Met Asp Arg165 170 175Glu Glu Val Glu Ile Thr Thr His Phe Arg Arg Lys Arg Arg Val Arg180 185 190Asp Asn Met Thr Lys Lys Met Val Thr Gln Arg Thr Ile Gly Lys Lys195 200 205Lys Gln Arg Leu Ser Lys Arg Ser Tyr Leu Ile Arg Ala Leu Thr Leu210 215 220Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala225 230 235 240Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu245 250 255Thr Leu Ala Arg Ser Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro260 265 270Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys275 280 285Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly290 295 300Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala305 310 315 320Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Ile325 330 335Leu Ser Met Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly340 345 350Lys Gly Tyr Met Phe Glu Ser Lys Arg Met Lys Leu Arg Thr Gln Ile355 360 365Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Glu Ser370 375 380Thr Arg Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Ile Asp Gly Thr385 390 395 400Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser405 410 415Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Lys Lys Tyr Thr420 425 430Arg Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala435 440 445Leu Ile Val Asn Ala Pro Asn Tyr Glu Gly Ile Gln Ala Gly Val Asn450 455 460Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys465 470 475 480Lys Ser Tyr Ile Asn Lys Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe485 490 495Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe500 505 510Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr515 520 525Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala530 535 540Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg545 550 555 560Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu565 570 575Lys Lys Leu Trp Asp Gln Thr Arg Ser Asn Ala Gly Leu Leu Val Ser580 585 590Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu595 600 605Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Arg Gly Arg Leu610 615 620Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Asp Ser Val625 630 635 640Asn Asn Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu645 650 655Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg660 665 670Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met675 680 685Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser690 695 700Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser705 710 715 720Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys725 730 735Lys Glu Glu Phe Ser Glu Ile Ile Arg Ile Cys Ser Thr Ile Glu Glu740 745 750Leu Arg Arg Gln Thr75516759PRTinfluenza A virus 16Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr1 5 10 15Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys20 25 30Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys35 40 45Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met50 55 60Asp Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys65 70 75 80Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val85 90 95Thr Trp Trp Asn Arg Asn Gly Pro Thr Ala Ser Thr Val His Tyr Pro100 105 110Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly115 120 125Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg130 135 140Val Asp Thr Asn Pro Gly His Ala Asp Leu Arg Ala Lys Glu Ala Gln145 150 155 160Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile165 170 175Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu180 185 190Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu195 200 205Lys Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr210 215 220Gly Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp225 230 235 240Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Val Asp245 250 255Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Ala Val260 265 270Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln275 280 285Ile Gly Gly Ile Arg Met Val Asp Ile Leu Arg Gln Asn Pro Thr Glu290 295 300Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser305 310 315 320Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser325 330 335Ser Val Lys Arg Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu340 345 350Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg355 360 365Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu370 375 380Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val385 390 395 400Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly405 410 415Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His420 425 430Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn435 440 445Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu450 455 460Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Ile Arg Val465 470 475 480Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val485 490 495Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Val Leu500 505 510Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Ala515 520 525Ile Thr Tyr Ser Ser Ser Met Met Trp Glu Ile Ser Gly Pro Glu Ser530 535 540Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ser Leu545 550 555 560Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Met Glu565 570 575Phe Glu Pro Phe Gln Ser Leu Val Pro Lys Ala Thr Arg Ser Arg Tyr580 585 590Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Ile Gly595 600 605Thr Phe Asp Thr Val Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala610 615 620Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val625 630 635 640Arg Gly Ser Gly Leu Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe645 650 655Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala660 665 670Gly Glu Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser675 680 685Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asp Lys Arg Tyr690 695 700Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Asn Leu Ala Lys Gly Glu705 710 715 720Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys725 730 735Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys740 745 750Arg Ile Arg Met Ala Ile Asn755171780DNAinfluenza A virus 17ttattcgtct tcaaggagca aaagcagggg ttataccata gacaaccgaa caaagacaat 60gaccatcact tttctcatcc tcctgttcac agtagtgaaa ggggaccaaa tatgcatcgg 120ataccatgcc aacaattcca cagaaaaagt tgacacaatc ttggaacgaa acgtcaccgt 180gactcatgcc aagaacattc ttgaaaagac gcataatgga aagttgtgca gattgagtgg 240aatccctcca ttggaactgg gggattgcag cattgcaggt tggctccttg gaaatccgga 300atgtgaccgg ctcttaagtg tacctgaatg gtcctatata gtggaaaagg aaaacccggt 360gaatggtctg tgctatccag gcagtttcaa tgattatgag gaattgaaac atcttctcac 420cagtgtgaca cactttgaga aagttaagat tctgcccaga gatcaatgga cccagcacac 480aacaactggt ggttctcggg cctgtgcagt atctggaaac ccgtcattct ttaggaacat 540ggtttggctt acaaagaaag ggtcaaacta ctcaattgct aaaaggtcat acaacaacac 600aagtggggag caaatgctgg taatatgggg gatacatcac cccaatgacg atgcggaaca 660gaggacactg taccagaatg tgggaacata tgtttccgtt ggaacatcaa cactaaataa 720gaggtcaatc cctgaaatag caacaaggcc caaagtcaat ggactgggag gaagaatgga 780attctcttgg actctattgg agacatggga tgtcataaat tttgagagca ctggtaattt 840aattgcacca gaatacggat tcaaaatatc aaagagagga agctcaggaa ttatgaagac 900agagaaaata cttgaaaatt gtgaaaccaa atgtcagacc cccttggggg caataaatac 960aacattgccc tttcacaaca ttcacccatt gacaataggt gagtgcccca agtatgtaaa 1020gtcagataga ctgattttgg cgacaggagt aagaaatgtc ccccagattg aatcaagggg 1080attgtttgga gcaatagctg ggtttataga aggcggatgg caagggatgg ttgatggctg 1140gtatgggtac catcacagca atgatcaagg atcaggatat gcagcagaca aagaatccac 1200tcaaaaggca attgatggga taactaacaa agtaaattct gtgattgaaa agatgaacac 1260tcagtttgag gctgttggga aagagttcaa caacctagag agaaggctgg aaaacttaaa 1320taaaaagatg gaagatggat ttattgatgt atggacatat aatgccgaac tcctagttct 1380aatggaaaat gagaggacac ttgatttcca tgattctaat gtgaagaatc tgtacgataa 1440ggtcagaatg caattgagag acaatgctaa ggaaataggg aacggatgct ttgagtttta 1500tcataaatgt gatgatgaat gcatgaatag tgtcaggaat gggacatatg attatcccaa 1560atatgaggaa gagtccaagc tgaacaggaa cgaaatcaaa ggagtgaaat tgagcaatat 1620gggggtttat caaatacttg ctatatacgc tacagttgca ggctctttgt cactggcaat 1680catgatagct gggatttctt tctggatgtg ttctaatggg tctctgcaat gcagaatttg 1740catatgactg taagtcaatt tgtaattaaa aaaactcctt 178018991DNAinfluenza A virus 18gtttaaagat gagtcttcta accgaggtcg aaacgtatgt tctctctatc gtcccgtcag 60gccccctcaa agccgagata gcacagagac tcgaagacgt ttttgcaggg aaaaacaccg 120atcttgaggc tctcatggaa tggctaaaga caagaccaat cctgtcacct ctgactaagg 180ggattttagg gtttgtgttc acgctcaccg tgcccagtga gcgaggactg cagcgtagac 240gttttgttca gaatgccctc aatgggaatg gtgacccgaa caacatggac aaggcggtca 300aactttacag gaaactaaaa agggaaataa cattccatgg ggccaaagaa gtagcgctca 360gttactctgc tggtgcactt gccagttgca tgggcctcat atacaacaga atgggaactg 420tcaccactga ggttgccttt ggtctggtat gcgcaacctg tgaacagatt gctgattctc 480agcatcgatc ccatagacaa atggtgacaa caaccaatcc actaatcagg cacgagaaca 540gaatggtgat agccagcaca acagctaaag caatggaaca aatggctgga tcaagcgaac 600aagcagcaga ggctatggag gttgccagcc aggctagaca aatggtacag gcaatgagaa 660caattgggac tcaccctagt tccagcactg gtctaaaaga tgatcttctt gaaaatttac 720aggcctatca gaaacggatg ggagtgcaaa tgcaacgatt caaatgatcc tctcactgat 780gctgcaagca tcattgggat tttgcacctg atattgtgga ttcttgatcg tctttttttc 840aaatgcattt accgtcgctt taaatacggt ctgcaaagag ggccttctac ggaaggagtg 900ccggagtcca tgagggaaga atatcgacag aaacagcaga gtgctgtgga tgttgacgat 960ggtcattttg tcaacatagt gctagagtaa a 991191438DNAinfluenza A virus 19agatgaatcc gaatcagaag ataataacaa tcggggtagt gaataccact ctgtcaacaa 60tagcccttct cattggagtg ggaaacttaa ttttcaacac agtcatacat gagaaaatag 120gagaccatca aatagtgacc tatccaacaa taacgacccc tgcagtaccg aactgcagtg 180acactataat aacatacaat aacactgtga taaacaacat aacaacaaca ataataactg 240aagaagaaag gcctttcaag tctccactac cgctgtgccc cttcagagga ttcttccctt 300ttcacaagga caatgcaata cgactgggtg aaaacaaaga cgtcatagtc acaagagagc 360cttatgttag ctgcgataat gacaactgct ggtcctttgc tctcacacaa ggagcattgc 420tagggaccaa acatagcaat gggaccatta aagacaggac accatatagg tctctaattc 480gtttcccaat aggaacagct ccagtactag gaaattataa agagatatgc attgcttggt 540cgagcagcag ttgctttgac gggaaagagt ggatgcatgt gtgcatgaca gggaacgata 600atgatgcaag tgcccagata atatatggag ggagaatgac agactccatt aaatcatgga 660gaaaggacat actaagaact caggagtctg aatgccaatg cattgacggg acttgtgttg 720ttgctgtcac agatggccct gctgctaata gtgcagatta cagggtttac tggatacggg 780agggaaaaat aataaagtat gaaaatgttc ccaaaacaaa gatacaacac ttagaagaat 840gttcctgcta tgtggacatt gatgtttact gtatatgtag ggacaattgg aagggctcta 900acagaccttg gatgagaatc aacaacgaga ctatactgga aacagggtat gtatgtagta 960aattccactc agacaccccc aggcccgctg acccttcaac aatgtcatgt gactccccaa 1020gcaatgtcaa tggaggaccc ggagtgaagg ggtttggttt caaagctggc gatgatgtat 1080ggttaggtag aacagtgtcg actagtggta gatcgggctt tgaaattatc aaagttacag 1140aagggtggat caactctcct aaccatgtca aatcaattac acaaacacta gtgtcaaaca 1200atgactggtc aggctattcc ggtagcttca ttgtcaaagc caaggactgt tttcagccct 1260gtttttatgt tgagcttata cgagggaggc ccaacaagaa tgatgacgtc tcttggacaa 1320gtaatagtat agttactttc tgtggactag acaatgaacc tggatcggga aattggccag 1380atggttctaa cattgggttt atgcccaagt aatagaaaaa agcaccttgt ttctacta 1438201536DNAinfluenza A virus 20gataatcact caatgagtga catcgaagcc atggcgtctc aaggcaccaa acgatcatat 60gaacaaatgg agactggtgg ggaacgccag gatgccacag aaatcagagc atctgtcgga 120agaatgattg gtggaatcgg gaaattctac atccaaatgt gcactgaact caaactcagt 180gactatgagg gacgactaat ccaaaatagc atgacaatag agagaatggt gctctctgct 240tttgatgaga gaagaaataa atacctagaa gagcatccca gtgcagggaa ggatcctaag 300aaaactggag gacccatata tagaagagta gacggaaagt ggatgagaga actcattctt 360tatgacaaag aagaaataag gagagtttgg cgccaagcaa acaatggtga agatgcaaca 420gctggtcttg ctcatatcat gatttggcac tccaatctga atgatgccac gtaccagaga 480acaagagcgc ttgttcgcac cggaatggat cccagaatgt gctctctaat gcaaggttca 540acacttccca gaaggtctgg ggccgcaggt gctgcagtga aaggagttgg aacagtagca 600atggaattaa tcagaatgat caaacgtggg atcaatgacc gaaacttctg gagaggtgaa 660aatggacgaa agacaaggat tgcatatgaa agaatgtgca atattctcaa gggaaaattt 720cagacagctg cccagagggc aatgatggat caagtgagag aaagtcggaa ccccggaaac 780gctgagattg aagatctcat tttcctggca cggtcagcac ttattctaag aggatcagtt 840gcacataagt cttgcctgcc tgcttgtgtg tatgggcttg cagtggcaag tgggcatgac 900tttgaaaggg aagggtattc actggtcggg atagacccat ttaaattact ccaaaacagt 960caagtgttca gcttgataag accaaatgaa aacccagctc acaagagtca attagtgtgg 1020atggcatgcc actctgctgc atttgaggat ctgagggtat caagtttcat cagagggaag 1080aaagtgattc caagaggaag gctctccaca agaggggttc agattgcttc aaatgagaat 1140gtggaagcca tggattccaa taccttagag ctgagaagca gatactgggc cataaggacc 1200agaagtggag gaaataccaa tcaacagaag gcatccgcgg gccagatcag tgtgcaaccc 1260acattctcag tgcaacggaa tctccctttt gaaagagcaa ccattatggc agctttcagc 1320gggaacaatg aaggacggac atccgatatg cgaacagaag ttataaggat gatggaaaat 1380gcaaagccag aagatttgtc cttccagggg cggggagtct tcgagctctc ggacgaaaaa 1440gcaacgagcc cgatcgtgcc ttcctttgac atgagtaatg aaggatctta tttcttcgga 1500gacaatgcag aggagtatga cagttgagga aaaata 153621855DNAinfluenza A virus 21caacgacata atggactcca ccactgtgtc aagctttcag gtagactgtt tcctttggca 60catccgcaaa cggtttgcag acaatggatt gggtgatgcc ccattccttg atcggctccg 120ccgagatcaa aagtctctaa aaggaagagg caacaccctt ggcctcgaca ttgaaacagc 180cactcttgtt gggaaacaaa ttgtggagtg gattttgaaa gaagaatcca gcgatacact 240taagatgact attgcatccg tacctacttc gcgctattta gctgacatga ccctcgagga 300aatatcacga gactggttaa tgctcatgcc taggcaaaag ataataggcc ctctttgtgt 360gcgaatggac caggcgatca tggaaaagaa catcatactg aaagcgaact tcagtgtgat 420ctttaaccga ttagagactt tgatactact aagggctttc actgaggagg gaacaatcgt 480tggagaaatt tcacaattac cttctcttcc aggacatact aatgaggatg tcaaaaatgc 540aattggggtc ctcatcggag gacttgaatg gaatggtaac acggttcgag gctctgaaaa 600tctacagaga ttcgcttgga gaaaccgtaa tgaggatggg agaccttcac tacctccaga 660gaagaaatga aaagtggcga gagcaattgg gacaaaaatt tgaggaaata aggtggttaa 720ttgaagaagt gcggcacaga ttgaaaacga cagagaatag ttttgaacaa ataacattca 780tgcaagcctt acaactactg cttgaagtag aacaagagat aaggactttc tcgtttcagc 840ttatttaatg ataac 855222232DNAinfluenza A virus 22accaaaaaaa tggaagactt tgtgcgacaa tgcttcaatc caatgatcgt cgagcttgcg 60gaaaaggcaa tgaaggaata tggggaagat ccaaaaatcg aaactaacaa attcgcagca 120atatgcactc acttggaagt atgtttcatg tattcggatt tccacttcat tgatgagcga 180ggcgaatcaa taattgtgga atctggtgat ccaaatgcat tactgaagca ccgatttgaa 240ataattgaag gaagagacag aacaatggcc

tggacagtgg tgaatagcat ctgcaacacc 300acaggggtcg agaagcctaa atttcttccg gatctgtatg attacaagga gaaccgattc 360attgaaattg gagtaacacg gagagaggtt catatatact acctagagaa agccaacaag 420ataaaatctg agaagacaca cattcacatc ttttcattta ctggagaaga aatggccacc 480aaagcagact acactcttga tgaagaaagc agggcaagaa tcaaaaccag gctattcact 540ataagacaag aaatggccag caggggtcta tgggattcct ttcgtcagtc tgaaagaggc 600gaagagacaa ttgaggaaag atttgaaatc acaggaacca tgcgtaggct tgccgaccaa 660agtctcccac cgaatttctc cagccttgaa aactttagag cctatgtgga tggattcgaa 720ccgaacggct gcattgaggg caagctttct caaatgtcaa aagaagtgaa tgccaggatt 780gagccattcc tgaagacaac accacgccct ctcaaattac ctgatgggcc cccttgctct 840cagcggtcaa aattcttgct gatggatgcc ttgaaactaa gcatcgaaga cccgagtcac 900gagggagagg gtataccact atacgatgca atcaaatgca tgaagacatt tttcggctgg 960aaagagccca acataatcaa accacatgag aaaggcataa atcccaatta ctttctggct 1020tggaagcaag tgctagcaga actccaggac cttgaaaatg aagagaagat cccaaagaca 1080aagaacatga agaaaacaag ccaattgaag tgggtgcttg gtgagaatat ggcaccagaa 1140aaagtggact ttgaggattg caaggacatt ggcgatctaa agcagtatga tagtgatgag 1200ccagagccta gatcgctggc aagctggatc cagagtgaat tcaataaggc atgtgaattg 1260accgactcga gctggataga acttgatgaa ataggagaag atgttgctcc gattgaacac 1320attgcaagta tgaggaggaa ctattttaca gcagaagtgt cccactgcag ggccactgaa 1380tacataatga aaggagtcta cataaataca gctctgctca atgcatcttg tgcagccatg 1440gatgacttcc agctgattcc aatgataagc aaatgtagaa caaaggaagg aagacggaaa 1500acaaacctgt atgggttcat cataaaagga agatctcatt tgaggaatga tactgatgtg 1560gtaaactttg tgagcatgga attttctctc actgacccga ggctagaacc acacaaatgg 1620gagaagtatt gtgttcttga aataggagat atgctcctga ggactgcaat aggccaagtg 1680tcaaggccca tgttcctgta cattagaacc aatgggacct ccaagatcaa gatgaaatgg 1740ggtatggaaa tgaggcgctg cctccttcaa tctcttcaac agattgagag catgattgag 1800gctgagtctt ctgtcaaaga aaaggacatg actaaggaat tctttgaaaa caagtcggaa 1860acgtggccaa ttggagaatc ccccagagga gtagaggaag gctctattgg gaaagtatgc 1920agaaccttac tggcaaagtc tgtattcaac agtctgtacg catctccaca acttgagggg 1980ttttcagctg aatcgagaaa attgcttctc attgttcagg cacttaggga caacctggaa 2040cctgggacct tcgatcttgg ggggctatac gaagcaattg aggagtgcct gattaatgat 2100ccctgggttt tgcttaatgc atcttggttc aactccttcc tcacacatgc actgaaatag 2160ttgtggcaat gctactattt gctatccata ctgtccaaaa aagtaccttg tttctactaa 2220tagaagagcg at 2232232336DNAinfluenza A virus 23gaatggatgt caatccgacc ctacttttcc taaaagttcc agcgcaaaat gccataagca 60ccacattccc ttatactgga gatcctccat acagccatgg aacaggaaca ggatacacca 120tggacacagt caacagaaca catcaatatt cagaaaaagg gaagtggacg ataaacacag 180agactggggc accccagctc aacccgattg atggaccact acctgaggat aatgaaccaa 240gtggatatgc acaaacagaa tgtgttctgg aggccatggc tttccttgaa gagtcccacc 300cagggatatt tgagaattca tgccttgaaa caatggaagt tgttcaacaa acaagggtgg 360ataaactaac tcaaggtcgc caaacttatg attggacatt aaacagaaat caaccagcag 420caactgcatt ggccaacacc atagaagttt ttagatcgaa tagtctaaca gccaatgagt 480caggaagact aatagatttc ctaaaggatg taatggaatc aatggataga gaagaaatgg 540agataacaac acactttcaa agaaaaagga gagtgagaga caacatgacc aagaagatgg 600tcacacaaag aacaatagga aagaaaaaac aaagattgag taagagaagt tatctaataa 660gagcacttac attgaatacg atgaccaaag atgcagagag aggcaaatta aaaagaaggg 720ctatcgcaac acccgggatg caaattagag ggttcgtgta ctttgttgag actttagcta 780ggagcatttg cgaaaagctt gaacagtctg gactcccagt agggggaaat gaaaagaagg 840ccaaattggc aaatgttgtg agaaaaatga tgactaattc acaagacaca gagctttctt 900tcacaatcac tggggacaat actaagtgga atgaaaatca aaatcctcga atgttcctgg 960cgatgattac atatatcacc cgaaatcaac ccgagtggtt cagaaacatc ctgagcatgg 1020cacccataat gttttcaaac aaaatggcaa gactaggaaa agggtacatg ttcgagagta 1080aaaggatgaa gctccgaaca caaataccag cagaaatgct agcaagcatt gatctgaagt 1140atttcaatga atcaacaagg aagaaaattg agaaaataag gcctcttcta atagatggca 1200cagcatcatt gagccctgga atgatgatgg gcatgttcaa catgctaagt acggttttgg 1260gagtctcgat actgaatctt ggacaaaaga aatacaccag gacaacatac tggtgggatg 1320ggctccaatc ctccgacgat tttgccctca ttgtgaatgc accaaattat gagggaatac 1380aagcaggagt gaatagattc tacaggacct gcaagttagt aggaataaac atgagcaaaa 1440agaagtccta tataaataaa acagggacat ttgaattcac aagctttttt tatcgctatg 1500ggtttgtggc taattttagc atggagctgc ccagttttgg agtgtctgga ataaatgaat 1560cagctgatat gagtattgga gtaacagtga taaagaacaa tatgataaac aatgatcttg 1620gacctgcaac agcccagatg gcccttcaat tgttcatcaa agactacaga tacacatata 1680ggtgccacag aggagacaca caaattcaga cgagaagatc attcgagcta aagaagctat 1740gggatcaaac ccgatcaaat gcaggactat tagtatctga tggaggacca aacttataca 1800atatcaggaa tcttcacatt cctgaagtct gcttaaaatg ggagctaatg gatgaggatt 1860atcggggaag gctttgtaat cccctgaatc cttttgtcag ccataaagag attgattctg 1920taaacaatgc tgtggtgatg ccagcccatg gcccagccaa aagcatggaa tatgatgccg 1980ttgcaactac acactcctgg atccccaaga ggaatcgctc tattctcaac acaagccaaa 2040ggggaattct tgaggatgaa cagatgtacc agaagtgctg caacctgttc gagaaatttt 2100tccctagtag ttcatacagg agaccggttg gaatttctag catggtggag gccatggtgt 2160ctagggcccg gattgatgcc agaatcgact tcgagtctgg acggattaag aaagaagagt 2220tctctgagat cataaggatc tgttccacca ttgaagaact cagacggcaa acatgatgaa 2280tttggcttgt ccttcatgaa aaaatgcctt gtttctacta ataccgagac gatata 2336242297DNAinfluenza A virus 24tatattcaat atggagagaa taaaagaact aagagatcta atgtcgcagt cccgcactcg 60cgaaatcctc accaagacca ctgtggacca tatggccata atcaaaaagt acacatcagg 120aaggcaagag aagaaccctg cactcagaat gaagtggatg atggcaatga agtacccaat 180cacagcagac aagagaataa tggacatgat tccagagaga aatgaacaag gacaaaccct 240ctggagcaaa acaaacgatg ctggatcgga ccgcgtgatg gtatcacccc tggctgtaac 300atggtggaat aggaatggcc caacagcaag cacagttcac taccctaagg tatataaaac 360ttatttcgaa aaagtcgaaa ggttaaaaca tggtaccttt ggccctgtcc acttcaggaa 420tcaagttaaa ataagaagga gagttgacac aaaccctggt cacgcagatc tcagtgccaa 480ggaggcacag gatgtgatca tggaagttgt tttcccaaat gaggtggggg caagaatact 540gacatcagag tcacagctga caataacaaa agagaagaaa gaagagctcc aggattgtaa 600aattgctccc ttaatggtgg catacatgct agaaaaagag ttggtccgta aaacgaggtt 660tctcccggtg gctggtggaa caggcagtgt ttatattgaa gtgctgcatt taactcaggg 720gacatgctgg gagcaaatgt acactccagg aggagaagtg agaaatgatg atgttgacca 780aagtttgatt attgctgcta gaaacatagt aagaagagca gcagtgtcag cagacccatt 840agcatctctt ttggaaatgt gccacagcac acagattgga ggaataagga tggtggacat 900ccttagacag aatccaacgg aggaacaagc cgtagacata tgcaaggcag caatggggct 960gaggattagc tcctctttca gctttggtgg gttcactttc aaaagaacaa gtgggtcatc 1020agttaagaga gaagaagaag tgctcacggg caaccttcaa acactgaaaa taagagtaca 1080tgaaggatat gaagaattca caatggtcgg gagaagagca acagctattc tcagaaaagc 1140aaccaggaga ttgatccagt taatagtaag tgggagagat gagcagtcaa ttgctgaggc 1200aataattgtg gccatggtat tttcacaaga ggattgcatg atcaaggcag ttaggggcga 1260tctgaacttt gtcaataggg caaaccagcg actgaatccc atgcatcaac tcttgaggca 1320tttccaaaaa gatgcaaaag tgcttttcca gaattgggga attgaaccca tcgacaatgt 1380gatgggaatg atcgggatat tgcccgatat gaccccaagc acggagatgt cgctgagagg 1440gataagagtc agcaaaatgg gagtagatga atactccagc acagagagag tggtagtgag 1500cattgacaga tttttgaggg ttcgggatca acgagggaac gtactattgt ctcctgaaga 1560ggtcagtgag acacaaggga cggagaaatt ggcaataact tattcgtcat cgatgatgtg 1620ggagatcagt ggccctgagt cagtgctggt caacacttat caatggatca taaggaattg 1680ggaaagtttg aaaattcaat ggtcacaaga tcccacgatg ttatacaaca aaatggaatt 1740tgaaccattt cagtctcttg tccctaaagc aaccagaagt cgttacagtg ggttcgtgag 1800gacactgttc cagcaaatgc gggatgtgat tggaacattt gacactgtcc aaataataaa 1860acttctcccc tttgctgctg ctccaccaga acagagtagg atgcagtttt cctcactgac 1920tgtgaatgtg agaggatcag ggttgaggat actggtaaga ggcaattctc cagtattcaa 1980ttacaataaa gcaaccaaaa ggcttacagt tcttggaaag gatgcaggtg aattgaccga 2040agacccagat gaaggcacag ctggagtgga gtctgctgtc ctgaggggat ttctcatttt 2100gggcaaagaa gacaagagat atggtccagc attaagcatc aatgaactga gcaatcttgc 2160aaaaggagag aaagctaatg tgctaattgg gcaaggagac gtagtgttgg taatgaaacg 2220gaaacgggac tctagcatac ttactgacag ccagacagcg accaaaagaa ttcggacggc 2280catcaattag tgtcgaa 229725562PRTinfluenza A virus 25Met Thr Ile Thr Phe Leu Ile Leu Leu Phe Thr Val Val Lys Gly Asp1 5 10 15Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val Asp20 25 30Thr Ile Leu Glu Arg Asn Val Thr Val Thr His Ala Lys Asn Ile Leu35 40 45Glu Lys Thr His Asn Gly Lys Leu Cys Arg Leu Ser Gly Ile Pro Pro50 55 60Leu Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro65 70 75 80Glu Cys Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile Val Glu85 90 95Lys Glu Asn Pro Val Asn Gly Leu Cys Tyr Pro Gly Ser Phe Asn Asp100 105 110Tyr Glu Glu Leu Lys His Leu Leu Thr Ser Val Thr His Phe Glu Lys115 120 125Val Lys Ile Leu Pro Arg Asp Gln Trp Thr Gln His Thr Thr Thr Gly130 135 140Gly Ser Arg Ala Cys Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn145 150 155 160Met Val Trp Leu Thr Lys Lys Gly Ser Asn Tyr Ser Ile Ala Lys Arg165 170 175Ser Tyr Asn Asn Thr Ser Gly Glu Gln Met Leu Val Ile Trp Gly Ile180 185 190His His Pro Asn Asp Asp Ala Glu Gln Arg Thr Leu Tyr Gln Asn Val195 200 205Gly Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys Arg Ser Ile210 215 220Pro Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Leu Gly Gly Arg Met225 230 235 240Glu Phe Ser Trp Thr Leu Leu Glu Thr Trp Asp Val Ile Asn Phe Glu245 250 255Ser Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys260 265 270Arg Gly Ser Ser Gly Ile Met Lys Thr Glu Lys Ile Leu Glu Asn Cys275 280 285Glu Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro290 295 300Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val305 310 315 320Lys Ser Asp Arg Leu Ile Leu Ala Thr Gly Val Arg Asn Val Pro Gln325 330 335Ile Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly340 345 350Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn355 360 365Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala370 375 380Ile Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn385 390 395 400Thr Gln Phe Glu Ala Val Gly Lys Glu Phe Asn Asn Leu Glu Arg Arg405 410 415Leu Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Ile Asp Val Trp420 425 430Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu435 440 445Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Met450 455 460Gln Leu Arg Asp Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe465 470 475 480Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser Val Arg Asn Gly Thr485 490 495Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu500 505 510Ile Lys Gly Val Lys Leu Ser Asn Met Gly Val Tyr Gln Ile Leu Ala515 520 525Ile Tyr Ala Thr Val Ala Gly Ser Leu Ser Leu Ala Ile Met Ile Ala530 535 540Gly Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile545 550 555 560Cys Ile26252PRTinfluenza A virus 26Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro1 5 10 15Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe20 25 30Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr35 40 45Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe50 55 60Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val65 70 75 80Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys Ala85 90 95Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala100 105 110Lys Glu Val Ala Leu Ser Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met115 120 125Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe130 135 140Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg145 150 155 160Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu165 170 175Asn Arg Met Val Ile Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met180 185 190Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln195 200 205Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser210 215 220Ser Ser Thr Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr225 230 235 240Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys245 25027469PRTinfluenza A virus 27Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Val Val Asn Thr Thr1 5 10 15Leu Ser Thr Ile Ala Leu Leu Ile Gly Val Gly Asn Leu Ile Phe Asn20 25 30Thr Val Ile His Glu Lys Ile Gly Asp His Gln Ile Val Thr Tyr Pro35 40 45Thr Ile Thr Thr Pro Ala Val Pro Asn Cys Ser Asp Thr Ile Ile Thr50 55 60Tyr Asn Asn Thr Val Ile Asn Asn Ile Thr Thr Thr Ile Ile Thr Glu65 70 75 80Glu Glu Arg Pro Phe Lys Ser Pro Leu Pro Leu Cys Pro Phe Arg Gly85 90 95Phe Phe Pro Phe His Lys Asp Asn Ala Ile Arg Leu Gly Glu Asn Lys100 105 110Asp Val Ile Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Asn Asp Asn115 120 125Cys Trp Ser Phe Ala Leu Thr Gln Gly Ala Leu Leu Gly Thr Lys His130 135 140Ser Asn Gly Thr Ile Lys Asp Arg Thr Pro Tyr Arg Ser Leu Ile Arg145 150 155 160Phe Pro Ile Gly Thr Ala Pro Val Leu Gly Asn Tyr Lys Glu Ile Cys165 170 175Ile Ala Trp Ser Ser Ser Ser Cys Phe Asp Gly Lys Glu Trp Met His180 185 190Val Cys Met Thr Gly Asn Asp Asn Asp Ala Ser Ala Gln Ile Ile Tyr195 200 205Gly Gly Arg Met Thr Asp Ser Ile Lys Ser Trp Arg Lys Asp Ile Leu210 215 220Arg Thr Gln Glu Ser Glu Cys Gln Cys Ile Asp Gly Thr Cys Val Val225 230 235 240Ala Val Thr Asp Gly Pro Ala Ala Asn Ser Ala Asp Tyr Arg Val Tyr245 250 255Trp Ile Arg Glu Gly Lys Ile Ile Lys Tyr Glu Asn Val Pro Lys Thr260 265 270Lys Ile Gln His Leu Glu Glu Cys Ser Cys Tyr Val Asp Ile Asp Val275 280 285Tyr Cys Ile Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Trp Met290 295 300Arg Ile Asn Asn Glu Thr Ile Leu Glu Thr Gly Tyr Val Cys Ser Lys305 310 315 320Phe His Ser Asp Thr Pro Arg Pro Ala Asp Pro Ser Thr Met Ser Cys325 330 335Asp Ser Pro Ser Asn Val Asn Gly Gly Pro Gly Val Lys Gly Phe Gly340 345 350Phe Lys Ala Gly Asp Asp Val Trp Leu Gly Arg Thr Val Ser Thr Ser355 360 365Gly Arg Ser Gly Phe Glu Ile Ile Lys Val Thr Glu Gly Trp Ile Asn370 375 380Ser Pro Asn His Val Lys Ser Ile Thr Gln Thr Leu Val Ser Asn Asn385 390 395 400Asp Trp Ser Gly Tyr Ser Gly Ser Phe Ile Val Lys Ala Lys Asp Cys405 410 415Phe Gln Pro Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Asn Lys420 425 430Asn Asp Asp Val Ser Trp Thr Ser Asn Ser Ile Val Thr Phe Cys Gly435 440 445Leu Asp Asn Glu Pro Gly Ser Gly Asn Trp Pro Asp Gly Ser Asn Ile450 455 460Gly Phe Met Pro Lys46528498PRTinfluenza A virus 28Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Gly1 5 10 15Gly Glu Arg Gln Asp Ala Thr Glu Ile Arg Ala Ser Val Gly Arg Met20 25 30Ile Gly Gly Ile Gly Lys Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys35 40 45Leu Ser Asp Tyr Glu Gly Arg Leu Ile Gln Asn Ser Met Thr Ile Glu50 55 60Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu65 70 75 80Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile85 90 95Tyr Arg Arg Val Asp Gly Lys Trp Met Arg Glu Leu Ile Leu Tyr Asp100 105 110Lys Glu Glu Ile Arg Arg Val Trp Arg Gln Ala Asn Asn Gly Glu Asp115 120 125Ala Thr Ala Gly Leu Ala His Ile Met Ile Trp His Ser Asn Leu Asn130 135 140Asp Ala Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp145 150 155 160Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser165

170 175Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Val Ala Met Glu180 185 190Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg195 200 205Gly Glu Asn Gly Arg Lys Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn210 215 220Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp225 230 235 240Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu245 250 255Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His260 265 270Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala Val Ala Ser Gly275 280 285His Asp Phe Glu Arg Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe290 295 300Lys Leu Leu Gln Asn Ser Gln Val Phe Ser Leu Ile Arg Pro Asn Glu305 310 315 320Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala325 330 335Ala Phe Glu Asp Leu Arg Val Ser Ser Phe Ile Arg Gly Lys Lys Val340 345 350Ile Pro Arg Gly Arg Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn355 360 365Glu Asn Val Glu Ala Met Asp Ser Asn Thr Leu Glu Leu Arg Ser Arg370 375 380Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Lys385 390 395 400Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg405 410 415Asn Leu Pro Phe Glu Arg Ala Thr Ile Met Ala Ala Phe Ser Gly Asn420 425 430Asn Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Val Ile Arg Met Met435 440 445Glu Asn Ala Lys Pro Glu Asp Leu Ser Phe Gln Gly Arg Gly Val Phe450 455 460Glu Leu Ser Asp Glu Lys Ala Thr Ser Pro Ile Val Pro Ser Phe Asp465 470 475 480Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr485 490 495Asp Ser29219PRTinfluenza A virus 29Met Asp Ser Thr Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp1 5 10 15His Ile Arg Lys Arg Phe Ala Asp Asn Gly Leu Gly Asp Ala Pro Phe20 25 30Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Lys Gly Arg Gly Asn35 40 45Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Leu Val Gly Lys Gln Ile50 55 60Val Glu Trp Ile Leu Lys Glu Glu Ser Ser Asp Thr Leu Lys Met Thr65 70 75 80Ile Ala Ser Val Pro Thr Ser Arg Tyr Leu Ala Asp Met Thr Leu Glu85 90 95Glu Ile Ser Arg Asp Trp Leu Met Leu Met Pro Arg Gln Lys Ile Ile100 105 110Gly Pro Leu Cys Val Arg Met Asp Gln Ala Ile Met Glu Lys Asn Ile115 120 125Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Asn Arg Leu Glu Thr Leu130 135 140Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Thr Ile Val Gly Glu Ile145 150 155 160Ser Gln Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn165 170 175Ala Ile Gly Val Leu Ile Gly Gly Leu Glu Trp Asn Gly Asn Thr Val180 185 190Arg Gly Ser Glu Asn Leu Gln Arg Phe Ala Trp Arg Asn Arg Asn Glu195 200 205Asp Gly Arg Pro Ser Leu Pro Pro Glu Lys Lys210 21530716PRTinfluenza A virus 30Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu1 5 10 15Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asp Pro Lys Ile Glu Thr20 25 30Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr35 40 45Ser Asp Phe His Phe Ile Asp Glu Arg Gly Glu Ser Ile Ile Val Glu50 55 60Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu65 70 75 80Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn85 90 95Thr Thr Gly Val Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr100 105 110Lys Glu Asn Arg Phe Ile Glu Ile Gly Val Thr Arg Arg Glu Val His115 120 125Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His130 135 140Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp145 150 155 160Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe165 170 175Thr Ile Arg Gln Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg180 185 190Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr195 200 205Gly Thr Met Arg Arg Leu Ala Asp Gln Ser Leu Pro Pro Asn Phe Ser210 215 220Ser Leu Glu Asn Phe Arg Ala Tyr Val Asp Gly Phe Glu Pro Asn Gly225 230 235 240Cys Ile Glu Gly Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Arg245 250 255Ile Glu Pro Phe Leu Lys Thr Thr Pro Arg Pro Leu Lys Leu Pro Asp260 265 270Gly Pro Pro Cys Ser Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu275 280 285Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu290 295 300Tyr Asp Ala Ile Lys Cys Met Lys Thr Phe Phe Gly Trp Lys Glu Pro305 310 315 320Asn Ile Ile Lys Pro His Glu Lys Gly Ile Asn Pro Asn Tyr Phe Leu325 330 335Ala Trp Lys Gln Val Leu Ala Glu Leu Gln Asp Leu Glu Asn Glu Glu340 345 350Lys Ile Pro Lys Thr Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp355 360 365Val Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys370 375 380Lys Asp Ile Gly Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Pro385 390 395 400Arg Ser Leu Ala Ser Trp Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu405 410 415Leu Thr Asp Ser Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val420 425 430Ala Pro Ile Glu His Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala435 440 445Glu Val Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr450 455 460Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Asp Phe465 470 475 480Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg485 490 495Lys Thr Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg500 505 510Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr515 520 525Asp Pro Arg Leu Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu530 535 540Ile Gly Asp Met Leu Leu Arg Thr Ala Ile Gly Gln Val Ser Arg Pro545 550 555 560Met Phe Leu Tyr Ile Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys565 570 575Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile580 585 590Glu Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr595 600 605Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser610 615 620Pro Arg Gly Val Glu Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu625 630 635 640Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu645 650 655Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu660 665 670Arg Asp Asn Leu Glu Pro Gly Thr Phe Asp Leu Gly Gly Leu Tyr Glu675 680 685Ala Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala690 695 700Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Lys705 710 71531757PRTinfluenza A virus 31Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn1 5 10 15Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His20 25 30Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln35 40 45Tyr Ser Glu Lys Gly Lys Trp Thr Ile Asn Thr Glu Thr Gly Ala Pro50 55 60Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser65 70 75 80Gly Tyr Ala Gln Thr Glu Cys Val Leu Glu Ala Met Ala Phe Leu Glu85 90 95Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu100 105 110Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr115 120 125Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala130 135 140Asn Thr Ile Glu Val Phe Arg Ser Asn Ser Leu Thr Ala Asn Glu Ser145 150 155 160Gly Arg Leu Ile Asp Phe Leu Lys Asp Val Met Glu Ser Met Asp Arg165 170 175Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg180 185 190Asp Asn Met Thr Lys Lys Met Val Thr Gln Arg Thr Ile Gly Lys Lys195 200 205Lys Gln Arg Leu Ser Lys Arg Ser Tyr Leu Ile Arg Ala Leu Thr Leu210 215 220Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala225 230 235 240Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu245 250 255Thr Leu Ala Arg Ser Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro260 265 270Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys275 280 285Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly290 295 300Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala305 310 315 320Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Ile325 330 335Leu Ser Met Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly340 345 350Lys Gly Tyr Met Phe Glu Ser Lys Arg Met Lys Leu Arg Thr Gln Ile355 360 365Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Glu Ser370 375 380Thr Arg Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Ile Asp Gly Thr385 390 395 400Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser405 410 415Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Lys Lys Tyr Thr420 425 430Arg Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala435 440 445Leu Ile Val Asn Ala Pro Asn Tyr Glu Gly Ile Gln Ala Gly Val Asn450 455 460Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys465 470 475 480Lys Ser Tyr Ile Asn Lys Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe485 490 495Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe500 505 510Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr515 520 525Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala530 535 540Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg545 550 555 560Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu565 570 575Lys Lys Leu Trp Asp Gln Thr Arg Ser Asn Ala Gly Leu Leu Val Ser580 585 590Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu595 600 605Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Arg Gly Arg Leu610 615 620Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Asp Ser Val625 630 635 640Asn Asn Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu645 650 655Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg660 665 670Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met675 680 685Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser690 695 700Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser705 710 715 720Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys725 730 735Lys Glu Glu Phe Ser Glu Ile Ile Arg Ile Cys Ser Thr Ile Glu Glu740 745 750Leu Arg Arg Gln Thr75532759PRTinfluenza A virus 32Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr1 5 10 15Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys20 25 30Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys35 40 45Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met50 55 60Asp Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys65 70 75 80Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val85 90 95Thr Trp Trp Asn Arg Asn Gly Pro Thr Ala Ser Thr Val His Tyr Pro100 105 110Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly115 120 125Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg130 135 140Val Asp Thr Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln145 150 155 160Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile165 170 175Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu180 185 190Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu195 200 205Lys Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr210 215 220Gly Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp225 230 235 240Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Val Asp245 250 255Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Ala Val260 265 270Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln275 280 285Ile Gly Gly Ile Arg Met Val Asp Ile Leu Arg Gln Asn Pro Thr Glu290 295 300Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser305 310 315 320Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser325 330 335Ser Val Lys Arg Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu340 345 350Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg355 360 365Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu370 375 380Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val385 390 395 400Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly405 410 415Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His420 425 430Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn435 440 445Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu450 455 460Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Ile Arg Val465 470 475 480Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val485 490 495Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Val Leu500 505 510Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Ala515 520 525Ile Thr Tyr Ser Ser Ser Met Met Trp Glu Ile Ser Gly Pro Glu Ser530 535 540Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ser Leu545 550 555 560Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Met Glu565 570 575Phe Glu Pro Phe Gln Ser Leu Val Pro Lys Ala Thr Arg Ser Arg Tyr580 585 590Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Ile Gly595 600 605Thr Phe Asp Thr Val Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala610 615 620Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn

Val625 630 635 640Arg Gly Ser Gly Leu Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe645 650 655Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala660 665 670Gly Glu Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser675 680 685Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asp Lys Arg Tyr690 695 700Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Asn Leu Ala Lys Gly Glu705 710 715 720Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys725 730 735Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys740 745 750Arg Ile Arg Thr Ala Ile Asn7553321DNAartificialoligonucleotide primer 33caggcagttt caatgattat g 213420DNAartificialoligonucleotide primer 34ccatcaattg ccttttgagt 203521DNAartificialoligonucleotide primer 35agcaaaagca ggtgcgagat g 213624DNAartificialoligonucleotide primer 36agtagaaaca aggtgctttt ttct 24371689DNAinfluenza A virus 37atgaccatca cttttctcat cctcctgttc acagtagtga aaggggacca aatatgcatc 60ggataccatg ccaacaattc cacagaaaaa gttgacacaa tcttggaacg aaacgtcacc 120gtgactcatg ccaagaacat tcttgaaaag acgcataatg gaaagttgtg cagattgagc 180gggatccctc cattggaact gggggattgc agcattgcag gttggctcct tggaaatccg 240gaatgtgacc ggctcttaag tgtacctgaa tggtcctata tagtggaaaa ggaaaacccg 300gtgaatggtc tgtgctatcc aggcagtttc aatgattatg aggaattgaa acatcttctc 360accagtgtga cacactttga gaaagttaag attctgccca gagatcaatg gacccagcac 420acaacaactg gtggttctcg ggcctgtgca gtatctggaa acccgtcatt ctttaggaac 480atggtttggc ttacaaagaa agggtcaaac tacccaattg ctaaaaggtc atacaacaac 540acaagtgggg agcaaatgct ggtaatatgg gggatacatc accccaatga cgatgcggaa 600cagaggacac tgtaccagaa tgtgggaaca tatgtttccg ttggaacatc aacactaaat 660aagaggtcaa tccctgaaat agcaacaagg cccaaagtca atggactggg aggaagaatg 720gaattctctt ggactctatt ggagacatgg gatgtcataa attttgagag cactggtaat 780ttaattgcac cagaatacgg attcaaaata tcaaagagag gaagctcagg aattatgaag 840acagagaaaa tacttgaaaa ttgtgaaacc aaatgtcaga cccccttggg ggcaataaat 900acaacattgc cctttcacaa cattcaccca ttgacaatag gtgagtgccc caagtatgta 960aagtcagata gactgatttt ggcgacagga ctaagaaatg tcccccagat tgaatcaagg 1020ggattgtttg gagcaatagc tgggtttata gaaggcggat ggcaagggat ggttgatggc 1080tggtatgggt accatcacag caatgatcaa ggatcaggat atgcagcaga caaagaatcc 1140actcaaaagg caattgatgg gataactaac aaagtaaatt ctgtgattga aaagatgaac 1200actcagtttg aggctgttgg gaaagagttc aacaacctag agagaagact ggaaaactta 1260aataaaaaga tggaagatgg atttattgat gtatggacat ataatgccga actcctagtt 1320ctaatggaaa atgagaggac acttgatttc catgattcta atgtgaagaa tctgtacgat 1380aaggtcagaa tgcaattgag agacaatgct aaggaaatag ggaacggatg ctttgagttt 1440tatcataaat gtgatgatga atgcatgaat agtgtcagga atgggacata tgattatatc 1500aaatatgagg aagagtccaa gctgaacagg aacgaaatca aaggagtgaa attgagcaat 1560atgggggttt atcaaatact tgctatatac gctacagttg caggctcttt gtcactggca 1620atcatgatag ctgggatttc tttctggatg tgttctaatg ggtctctgca atgcagaatt 1680tgcatatga 1689381689DNAinfluenza A virus 38atgaccatca cttttctcat cctcctgttc acagtagtga aaggggacca aatatgcatc 60ggataccatg ccaacaattc cacagaaaaa gttgacacaa tcttggaacg aaacgtcacc 120gtgactcatg ccaagaacat tcttgaaaag acgcataatg gaaagttgtg cagattgagt 180ggaatccctc cattggaact gggggattgc agcattgcag gttggctcct tggaaatccg 240gaatgtgacc ggctcttaag tgtacctgaa tggtcctata tagtggaaaa ggaaaacccg 300gtgaatggtc tgtgctatcc aggcagtttc aatgattatg aggaattgaa acatcttctc 360accagtgtga cacactttga gaaagttaag attctgccca gagatcaatg gacccagcac 420acaacaactg gtggttctcg ggcctgtgca gtatctggaa acccgtcatt ctttaggaac 480atggtttggc ttacaaagaa agggtcaaac tactcaattg ctaaaaggtc atacaacaac 540acaagtgggg agcaaatgct ggtaatatgg gggatacatc accccaatga cgatgcggaa 600cagaggacac tgtaccagaa tgtgggaaca tatgtttccg ttggaacatc aacactaaat 660aagaggtcaa tccctgaaat agcaacaagg cccaaagtca atggactggg aggaagaatg 720gaattctctt ggactctatt ggagacatgg gatgtcataa attttgagag cactggtaat 780ttaattgcac cagaatacgg attcaaaata tcaaagagag gaagctcagg aattatgaag 840acagagaaaa tacttgaaaa ttgtgaaacc aaatgtcaga cccccttggg ggcaataaat 900acaacattgc cctttcacaa cattcaccca ttgacaatag gtgagtgccc caagtatgta 960aagtcagata gactgatttt ggcgacagga gtaagaaatg tcccccagat tgaatcaagg 1020ggattgtttg gagcaatagc tgggtttata gaaggcggat ggcaagggat ggttgatggc 1080tggtatgggt accatcacag caatgatcaa ggatcaggat atgcagcaga caaagaatcc 1140actcaaaagg caattgatgg gataactaac aaagtaaatt ctgtgattga aaagatgaac 1200actcagtttg aggctgttgg gaaagagttc aacaacctag agagaaggct ggaaaactta 1260aataaaaaga tggaagatgg atttattgat gtatggacat ataatgccga actcctagtt 1320ctaatggaaa atgagaggac acttgatttc catgattcta atgtgaagaa tctgtacgat 1380aaggtcagaa tgcaattgag agacaatgct aaggaaatag ggaacggatg ctttgagttt 1440tatcataaat gtgatgatga atgcatgaat agtgtcagga atgggacata tgattatccc 1500aaatatgagg aagagtccaa gctgaacagg aacgaaatca aaggagtgaa attgagcaat 1560atgggggttt atcaaatact tgctatatac gctacagttg caggctcttt gtcactggca 1620atcatgatag ctgggatttc tttctggatg tgttctaatg ggtctctgca atgcagaatt 1680tgcatatga 1689391410DNAinfluenza A virus 39atgaatccga atcagaagat aataacaatc ggggtagtga ataccactct gtcaacaata 60gcccttctca ttggagtggg aaacttagtt ttcaacacag tcatacatga gaaaatagga 120gaccatcaaa tagtgaccta tccaataata acgacccctg cagtaccgaa ctgcagtgac 180actataataa catacaataa cactgtgata aacaacataa caacaacaat aataactgaa 240gaagaaaggc ctttcaagtc tccactaccg ctgtgcccct tcagaggatt cttccctttt 300cacaaggaca atgcaatacg actgggtgaa aacaaagacg tcatagtcac aagagagcct 360tatgttagct gcgataatga caactgctgg tcctttgctc tcgcacaagg agcattgcta 420gggaccaaac atagcaatgg gaccattaaa gacaggacac catataggtc tctaattcgt 480ttcccaatag gaacagctcc agtactagga aattataaag agatatgcat tgcttggtcg 540agcagcagtt gctttgacgg gaaagagtgg atgcatgtgt gcatgacagg gaacgataat 600gatgcaagtg cccagataat atatggaggg agaatgacag actccattaa atcatggaga 660aaggacatac taagaactca ggagtctgaa tgccaatgca ttgacgggac ttgtgttgtt 720gctgtcacag atggccctgc tgctaatagt gcagattaca gggtttactg gatacgggag 780ggaaaaataa taaagtatga aaatgttccc aaaacaaaga tacaacactt agaagaatgt 840tcctgctatg tggacattga tgtttactgt atatgtaggg acaattggaa gggctctaac 900agaccttgga tgagaatcaa caacgagact atactggaaa cagggtatgt atgtagtaaa 960ttccactcag acacccccag gccagctgac ccttcaacaa tgtcatgtga ctccccaagc 1020aatgtcaatg gaggacccgg agtgaagggg tttggtttca aagctggcga tgatgtatgg 1080ttaggtagaa cagtgtcgac tagtggtaga tcgggctttg aaattatcaa agttacagaa 1140gggtggatca actctcctaa ccatgtcaaa tcaattacac aaacactagt gtccaacaat 1200gactggtcag gctattccgg tagcttcatt gtcaaagcca aggactgttt tcagccctgt 1260ttttatgttg agcttatacg agggaggccc aacaagaatg atgacgtctc ttggacaagt 1320aatagtatag ttactttctg tggactagac aatgaacctg gatcgggaaa ttggccagat 1380ggttctaaca ttgggtttat gcccaagtaa 1410401410DNAinfluenza A virus 40atgaatccga atcagaagat aataacaatc ggggtagtga ataccactct gtcaacaata 60gcccttctca ttggagtggg aaacttaatt ttcaacacag tcatacatga gaaaatagga 120gaccatcaaa tagtgaccta tccaacaata acgacccctg cagtaccgaa ctgcagtgac 180actataataa catacaataa cactgtgata aacaacataa caacaacaat aataactgaa 240gaagaaaggc ctttcaagtc tccactaccg ctgtgcccct tcagaggatt cttccctttt 300cacaaggaca atgcaatacg actgggtgaa aacaaagacg tcatagtcac aagagagcct 360tatgttagct gcgataatga caactgctgg tcctttgctc tcacacaagg agcattgcta 420gggaccaaac atagcaatgg gaccattaaa gacaggacac catataggtc tctaattcgt 480ttcccaatag gaacagctcc agtactagga aattataaag agatatgcat tgcttggtcg 540agcagcagtt gctttgacgg gaaagagtgg atgcatgtgt gcatgacagg gaacgataat 600gatgcaagtg cccagataat atatggaggg agaatgacag actccattaa atcatggaga 660aaggacatac taagaactca ggagtctgaa tgccaatgca ttgacgggac ttgtgttgtt 720gctgtcacag atggccctgc tgctaatagt gcagattaca gggtttactg gatacgggag 780ggaaaaataa taaagtatga aaatgttccc aaaacaaaga tacaacactt agaagaatgt 840tcctgctatg tggacattga tgtttactgt atatgtaggg acaattggaa gggctctaac 900agaccttgga tgagaatcaa caacgagact atactggaaa cagggtatgt atgtagtaaa 960ttccactcag acacccccag gcccgctgac ccttcaacaa tgtcatgtga ctccccaagc 1020aatgtcaatg gaggacccgg agtgaagggg tttggtttca aagctggcga tgatgtatgg 1080ttaggtagaa cagtgtcgac tagtggtaga tcgggctttg aaattatcaa agttacagaa 1140gggtggatca actctcctaa ccatgtcaaa tcaattacac aaacactagt gtcaaacaat 1200gactggtcag gctattccgg tagcttcatt gtcaaagcca aggactgttt tcagccctgt 1260ttttatgttg agcttatacg agggaggccc aacaagaatg atgacgtctc ttggacaagt 1320aatagtatag ttactttctg tggactagac aatgaacctg gatcgggaaa ttggccagat 1380ggttctaaca ttgggtttat gcccaagtaa 1410411772DNAinfluenza A virus 41agcaaaagca ggggttatac catagacaac cgaacaaaga caatgaccat cacttttctc 60atcctcctgt tcacagtagt gaaaggggac caaatatgta tcggatacca tgccaacaat 120tccacagaaa aagttgacac aatcttggaa cgaaacgtca ccgtgactca tgccaaggac 180attcttgaaa aaacgcataa tggaaagttg tgcagattaa gcgggatccc tccactggaa 240ctgggggatt gcagcattgc aggttggctc cttggaaatc cggaatgtga ccggctctta 300agtgtacctg aatggtccta tatagtggaa aaggaaaacc cggtgaatgg tctgtgctac 360ccaggcagtt tcaatgatta tgaggaattg aaacatctcc tcaccagtgt gacacacttt 420gagaaagtta agattctgcc cagagatcaa tggactcagc acacaacaac tggtggttct 480cgggcctgtg cagtgtctgg aaacccgtca ttctttagga acatggtttg gcttacaaag 540aaggggtcaa actacccaat tgctaaaagg tcatacaaca acacaagtgg ggagcaaatg 600ctggtaatct gggggataca tcatcccaat gacgatgcgg aacaaaggac actgtaccag 660aatgtgggaa catatgtttc cgttgggaca tcaacactaa ataagaggtc aatccctgaa 720atagcaacaa ggcccaaagt caatggacaa ggagggagaa tggaattctc ttggactcta 780ttggagacat gggatgtcat aaattttgag agcactggta atttaattgc accagaatac 840ggattcaaaa tatcaaagag aggaagctca ggaattatga agacagagaa aacacttgaa 900aattgtgaaa ccaaatgtca gacccccttg ggggcaataa atacaacatt gccctttcac 960aacattcacc cattgacaat aggtgagtgc cccaagtatg taaagtcaga cagactggtt 1020ttggcaacag gactaagaaa tgtccctcag attgaatcaa ggggattgtt tggagcaata 1080gctgggttta tagaaggcgg atggcaaggg atggttgatg gctggtatgg gtatcatcac 1140agcaatgatc aaggatcagg atatgcagca gacaaagaat ccactcaaaa ggcaattgat 1200gggataacta acaaagtaaa ttctgtgatt gaaaagatga acactcagtt tgaggctgtt 1260gggaaagagt tcaacaatct agagagaaga ctagaaaact taaataaaaa gatggaagat 1320ggatttcttg atgtatggac atataatgcc gaactcctag ttctaatgga gaatgagagg 1380acacttgatt tccatgactc taatgtgaag aatctgtacg ataaggtcag aatgcaatta 1440agagacaatg ctaaggaaat agggaacgga tgctttgagt tttatcataa atgtgatgat 1500gaatgcatga atagtgtcag gaatggaaca tatgattatc ccaaatatga ggaagagtcc 1560aagctgaaca ggaacgaaat aaaaggactg aaattgagca atatgggggt ctatcaaata 1620cttgctatat acgctacagt tgcaggctcc ttgtcactgg caatcatgat agctgggatt 1680tctttctgga tgtgttctaa tgggtctctg caatgcagaa tttgcatatg actgtaagtc 1740aatttgtaat taaaaacacc cttgtttcta ct 177242562PRTinfluenza A virus 42Met Thr Ile Thr Phe Leu Ile Leu Leu Phe Thr Val Val Lys Gly Asp1 5 10 15Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val Asp20 25 30Thr Ile Leu Glu Arg Asn Val Thr Val Thr His Ala Lys Asp Ile Leu35 40 45Glu Lys Thr His Asn Gly Lys Leu Cys Arg Leu Ser Gly Ile Pro Pro50 55 60Leu Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro65 70 75 80Glu Cys Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile Val Glu85 90 95Lys Glu Asn Pro Val Asn Gly Leu Cys Tyr Pro Gly Ser Phe Asn Asp100 105 110Tyr Glu Glu Leu Lys His Leu Leu Thr Ser Val Thr His Phe Glu Lys115 120 125Val Lys Ile Leu Pro Arg Asp Gln Trp Thr Gln His Thr Thr Thr Gly130 135 140Gly Ser Arg Ala Cys Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn145 150 155 160Met Val Trp Leu Thr Lys Lys Gly Ser Asn Tyr Pro Ile Ala Lys Arg165 170 175Ser Tyr Asn Asn Thr Ser Gly Glu Gln Met Leu Val Ile Trp Gly Ile180 185 190His His Pro Asn Asp Asp Ala Glu Gln Arg Thr Leu Tyr Gln Asn Val195 200 205Gly Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys Arg Ser Ile210 215 220Pro Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Gln Gly Gly Arg Met225 230 235 240Glu Phe Ser Trp Thr Leu Leu Glu Thr Trp Asp Val Ile Asn Phe Glu245 250 255Ser Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys260 265 270Arg Gly Ser Ser Gly Ile Met Lys Thr Glu Lys Thr Leu Glu Asn Cys275 280 285Glu Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro290 295 300Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val305 310 315 320Lys Ser Asp Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Val Pro Gln325 330 335Ile Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly340 345 350Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn355 360 365Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala370 375 380Ile Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn385 390 395 400Thr Gln Phe Glu Ala Val Gly Lys Glu Phe Asn Asn Leu Glu Arg Arg405 410 415Leu Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp420 425 430Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu435 440 445Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Met450 455 460Gln Leu Arg Asp Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe465 470 475 480Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser Val Arg Asn Gly Thr485 490 495Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu500 505 510Ile Lys Gly Leu Lys Leu Ser Asn Met Gly Val Tyr Gln Ile Leu Ala515 520 525Ile Tyr Ala Thr Val Ala Gly Ser Leu Ser Leu Ala Ile Met Ile Ala530 535 540Gly Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile545 550 555 560Cys Ile431428DNAinfluenza A virus 43tgcgagatga atccgaatca gaagataata acaatcgggg tagtgaatac cactctgtca 60acaatagccc ttctcattgg agtgggaaac ttagttttca acacagtcat acatgagaaa 120ataggagacc atcaaatagt gacccatcca acaataacga cccctgcagt accgaactgc 180agtgacacta taataacata caataacact gtgataaaca acataacaac aacaataata 240actgaagcag aaaggccttt caagtctcca ctaccgctgt gccccttcaa aggattcttc 300ccttttcaca aggacaatgc aatacgactg ggtgagaaca aagacgtcat agtcacaagg 360gagccttatg ttagctgcga taatgacaac tgctggtcct ttgctctcgc acaaggagca 420ttgctaggga ctaaacatag caatgggacc attaaagaca ggacaccata taggtctcta 480attcgtttcc caataggaac agctccagta ctgggaaatt acaaagagat atgcattgct 540tggtcgagca gcagttgctt tgacgggaaa gagtggatgc atgtgtgcat gacagggaac 600gataatgatg caagtgccca gataatatat ggagggagaa tgacagactc cattaaatca 660tggagaaagg acatactaag aacccaggag tctgaatgtc aatgcattga cgggacttgt 720gttgttgctg tcacagatgg ccctgctgct aatagtgcag accacagggt ttactggata 780cgggagggaa gaataataaa gtatgaaaat gttcccaaaa caaagataca acacttagaa 840gaatgttcct gctatgtgga cattgatgtt tactgtatat gtagggacaa ttggaagggc 900tctaacagac cttggatgag aatcaacaac gagactatac tggaaacagg gtatgtatgt 960agtaaattcc actcagacac ccccaggcca gctgaccctt caacaatgtc atgtgactcc 1020ccaagcaatg tcaatggagg acccggagtg aaggggtttg gtttcaaagc tggcaatgat 1080gtatggttag gtagaacagt gtcaactagt ggtagatcgg gctttgaaat tatcaaagtt 1140acagaagggt ggatcaactc tcctaaccat gtcaaatcaa ttacacaaac actagtgtcc 1200aacaatgact ggtcaggcta ttcaggtagc ttcattgtca aagccaagga ctgttttcag 1260ccctgttttt atgttgagct tatacgaggg aggcccaaca agaatgatga cgtctcttgg 1320acaagtaata gtatagttac tttctgtgga ctagacaatg aacctggatc gggaggttgg 1380ccggatggtt ctaacattgg gtttgtgccc aagtaataga aaaaagca 142844469PRTinfluenza A virus 44Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Val Val Asn Thr Thr1 5 10 15Leu Ser Thr Ile Ala Leu Leu Ile Gly Val Gly Asn Leu Val Phe Asn20 25 30Thr Val Ile His Glu Lys Ile Gly Asp His Gln Ile Val Thr His Pro35 40 45Thr Ile Thr Thr Pro Ala Val Pro Asn Cys Ser Asp Thr Ile Ile Thr50 55 60Tyr Asn Asn Thr Val Ile Asn Asn Ile Thr Thr Thr Ile Ile Thr Glu65 70 75 80Ala Glu Arg Pro Phe Lys Ser Pro Leu Pro Leu Cys Pro Phe Lys Gly85 90 95Phe Phe Pro Phe His Lys Asp Asn Ala Ile Arg Leu Gly Glu Asn Lys100 105 110Asp Val Ile Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Asn Asp Asn115 120 125Cys Trp Ser Phe Ala Leu Ala Gln Gly Ala Leu Leu Gly Thr Lys His130 135 140Ser Asn Gly Thr Ile Lys Asp Arg Thr Pro Tyr Arg Ser Leu Ile Arg145

150 155 160Phe Pro Ile Gly Thr Ala Pro Val Leu Gly Asn Tyr Lys Glu Ile Cys165 170 175Ile Ala Trp Ser Ser Ser Ser Cys Phe Asp Gly Lys Glu Trp Met His180 185 190Val Cys Met Thr Gly Asn Asp Asn Asp Ala Ser Ala Gln Ile Ile Tyr195 200 205Gly Gly Arg Met Thr Asp Ser Ile Lys Ser Trp Arg Lys Asp Ile Leu210 215 220Arg Thr Gln Glu Ser Glu Cys Gln Cys Ile Asp Gly Thr Cys Val Val225 230 235 240Ala Val Thr Asp Gly Pro Ala Ala Asn Ser Ala Asp His Arg Val Tyr245 250 255Trp Ile Arg Glu Gly Arg Ile Ile Lys Tyr Glu Asn Val Pro Lys Thr260 265 270Lys Ile Gln His Leu Glu Glu Cys Ser Cys Tyr Val Asp Ile Asp Val275 280 285Tyr Cys Ile Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Trp Met290 295 300Arg Ile Asn Asn Glu Thr Ile Leu Glu Thr Gly Tyr Val Cys Ser Lys305 310 315 320Phe His Ser Asp Thr Pro Arg Pro Ala Asp Pro Ser Thr Met Ser Cys325 330 335Asp Ser Pro Ser Asn Val Asn Gly Gly Pro Gly Val Lys Gly Phe Gly340 345 350Phe Lys Ala Gly Asn Asp Val Trp Leu Gly Arg Thr Val Ser Thr Ser355 360 365Gly Arg Ser Gly Phe Glu Ile Ile Lys Val Thr Glu Gly Trp Ile Asn370 375 380Ser Pro Asn His Val Lys Ser Ile Thr Gln Thr Leu Val Ser Asn Asn385 390 395 400Asp Trp Ser Gly Tyr Ser Gly Ser Phe Ile Val Lys Ala Lys Asp Cys405 410 415Phe Gln Pro Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Asn Lys420 425 430Asn Asp Asp Val Ser Trp Thr Ser Asn Ser Ile Val Thr Phe Cys Gly435 440 445Leu Asp Asn Glu Pro Gly Ser Gly Gly Trp Pro Asp Gly Ser Asn Ile450 455 460Gly Phe Val Pro Lys465

Claims

1. An isolated biologically active compound comprising an amino acid sequence, wherein the amino acid sequence of the compound and the amino acid sequence of SEQ ID NO:21 have at least 80% identity.

2. The isolated biologically active compound of claim 1 wherein the polypeptide comprises at least one conservative substitution of the amino acid sequence of SEQ ID NO:21.

3. The isolated biologically active compound of claim 1 wherein the compound comprises the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe.

4. The isolated biologically active compound of claim 3 wherein the compound inhibits growth of an E. Coli, a Serratia proteus, or a Salmonella spp.

5. The isolated biologically active compound of claim 1 wherein the compound inhibits growth of a Gram positive microbe Lactobacillus spp., Lactococcus spp., a Streptococcus spp., a Staphylococcus spp., or a Bacillus spp.

6. The isolated biologically active compound of claim 1 wherein the compound is produced by a Bifidobacterium.

7. A composition comprising the isolated biologically active compound of claim 1 and a food product.

8. A composition comprising the isolated biologically active compound of claim 1 and a pharmaceutically acceptable carrier.

9. An isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:21 have at least 80% identity, or (b) the full complement of the nucleotide sequence of (a).

10. The isolated polynucleotide of claim 9 wherein the nucleotide sequence encodes a polypeptide comprising the amino acid sequence SEQ ID NO:21.

11. A polynucleotide comprising the isolated polynucleotide of claim 9 operably linked to a heterologous regulatory sequence.

12. A vector comprising the isolated polynucleotide of claim 9.

13. A cell comprising the isolated polynucleotide of claim 9.

14. An isolated lantibiotic, wherein the lantibiotic inhibits growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe.

15. The lantibiotic of claim 14 wherein the lantibiotic comprises an amino acid sequence, wherein the amino acid sequence of the compound and the amino acid sequence of SEQ ID NO:21 have at least 80% identity.

16. The isolated lantibiotic of claim 15 wherein the Gram negative microbe is an E. coli, a Serratia proteus, or a Salmonella spp.

17. A composition comprising a lantibiotic and a food product, wherein the lantibiotic comprises the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe.

18. The composition of claim 17 wherein the lantibiotic is present on the surface of the food product.

19. The composition of claim 17 wherein the lantibiotic is present in the food product.

20. The composition of claim 17 wherein the lantibiotic comprises an amino acid sequence, wherein the amino acid sequence of the lantibiotic and the amino acid sequence of SEQ ID NO:21 have at least 80% identity.

21. The composition of claim 17 wherein the Gram negative microbe is an E. coli, a Serratia proteus, or a Salmonella spp.

22. A composition comprising a lantibiotic and a pharmaceutically acceptable carrier, wherein the lantibiotic comprises the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe.

23. A method for producing a lantibiotic, comprising:growing an isolated Bifidobacterium under conditions suitable for producing the lantibiotic, wherein the Bifidobacterium produces a lantibiotic.

24. The method of claim 23 wherein the growing comprises growing the Bifidobacterium on a surface.

25. The method of claim 23 wherein the Bifidobacterium is B. longum.

26. A lantibiotic produced by the process of claim 23.

27. A method for producing a lantibiotic, comprising:growing a microbe comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:21 have at least 80% identity, wherein the microbe is grown under conditions suitable for producing the polypeptide, and wherein the microbe produces the polypeptide.

28. The method of claim 27 wherein the microbe further comprises a polynucleotide encoding a polypeptide selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or a combination thereof.

29. The method of claim 27 wherein the growing comprises growing the microbe on a surface.

30. The method of claim 27 wherein the microbe is a Bifidobacterium spp.

31. The method of claim 27 wherein the isolating comprises extraction with a composition comprising an alcohol.

32. The method of claim 31 wherein the alcohol is methanol.

33. A method of using a lantibiotic comprising adding the lantibiotic to a food product, wherein the lantibiotic comprises the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe.

34. The method of claim 32 wherein the adding comprises applying the lantibiotic to the surface of the food product.

35. The method of claim 34 wherein the lantibiotic is applied by bringing a surface of a casing, film, or packaging material comprising the lantibiotic into contact with the food product.

36. The method of claim 33 wherein the adding comprises adding the lantibiotic to the food product.

37. The method of claim 33 wherein the lantibiotic is a food preservative.

38. A dentifrice comprising a biologically active compound comprising an amino acid sequence, wherein the amino acid sequence of the compound and the amino acid sequence of SEQ ID NO:21 have at least 80% identity, wherein the compound comprises the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe.

39. The dentifrice of claim 38 wherein the dentifrice is a mouthwash or a toothpaste.

40. A method of using a lantibiotic comprising administering a composition comprising a lantibiotic to an animal, wherein the subject has or is at risk of an infection by a microbe that is inhibited by the lantibiotic, and wherein the lantibiotic comprises the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe.

41. The method of claim 38 wherein the composition is administered topically.

42. The method of claim 38 wherein the composition comprises a pharmaceutically acceptable carrier.

43. The method of claim 40 wherein the animal is a human.

44. An isolated biologically active polypeptide, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:2 have at least 80% identity.

45. An isolated polynucleotide encoding the polypeptide of claim 44.

46. A composition comprising a Bifidobacterium that produces a lantibiotic, wherein the lantibiotic comprises the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe.

47. The composition of claim 46 wherein the Bifidobacterium is encapsulated.

48. The composition of claim 46 wherein the composition further comprises a food product.

49. A method comprising administering a Bifidobacterium to an animal in need thereof, wherein the Bifidobacterium will produce a lantibiotic that comprises the characteristic of inhibiting growth of a Gram negative microbe in conditions that do not damage the outer membrane of the Gram negative microbe.

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